Pluripotent Stem Cell-Engineered Immune Cells for Off-the-Shelf Cell Therapy

Abstract

Embodiments of the present disclosure include compositions and methods relating to engineered monoclonal TCR-armed genetically engineered T (TARGET) cells for off-the-shelf use in clinical therapy. In certain embodiments, TARGET cells are produced from pluripotent stem cell lines and therefore can be in unlimited supply. TARGET cell products can be conventional αβ T cells, γδ T cells, and invariant natural killer T (iNKT) cells, non-invariant natural killer T (NKT) cells, mucosal-associated invariant T (MAIT) cells, and other non-conventional T cells. TARGET cell products can also be engineered to be CD8-positive or CD4-positive T cell subtypes, and CD4-positive TARGET cells can be engineered to be various T H It can further be designed to be a polarized subtype.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit under 35 U.S.C. Section 119(e) of co-pending and commonly assigned U.S. Provisional Patent Application No. 63 / 485,700, entitled "PLURIPOTENT STEM CELL-ENGINEERED IMMUNE CELLS FOR OFF-THE-SHELF CELL THERAPY," filed February 17, 2023, which is incorporated herein by reference.

[0002] Technical Field Embodiments of the present disclosure relate to the fields of immunology, cell biology, molecular biology, and medicine, including at least cancer medicine. [Background technology]

[0003] background Cancer affects tens of millions of people worldwide and is a major threat to public health in the United States. It is predicted that there will be more than 2 million new cancer cases and more than 600,000 cancer deaths in the United States in 2024. Despite existing treatments, cancer patients continue to suffer from the ineffectiveness of these treatments, their toxicity, and the risk of recurrence. Therefore, novel therapies for cancer are desperately needed. Over the past decade, immunotherapy has become a new generation of cancer medicine. In particular, cell-based therapies have shown great promise. A notable example is chimeric antigen receptor (CAR)-engineered adoptive T-cell therapy, which targets certain hematological cancers with impressive efficacy.

[0004] However, most current treatment protocols consist of autologous adoptive cell transfer, in which immune cells collected from a patient are manufactured and used to treat that single patient. Such approaches are costly, labor-intensive to manufacture, and difficult to widely distribute to all patients in need. Therefore, there is a great need for allogeneic immune cell products that can be manufactured on a large scale and easily distributed to treat a larger number of patients.

[0005] Despite existing treatment methods, cancer patients still suffer from the ineffectiveness of these treatments, their toxicity and the risk of recurrence.Therefore, there is a dire need for new treatment methods for diseases such as cancer and autoimmune disease.The present disclosure provides a solution to the long-standing need for these treatment methods, but also provides a treatment method that can be delivered or distributed more widely. Summary of the Invention [Means for solving the problem]

[0006] A brief summary Embodiments are provided to address the need for new therapeutic approaches, and more specifically, the need for cell therapies that are not hindered by the challenges posed by using autologous cells to personalize therapy. Embodiments of the present invention include methods designed to use pluripotent stem cell (PSC) cells to generate populations of selected immune cells for "off-the-shell" use. The ability to manufacture "off-the-shell" therapeutic cell populations or cell populations that can be used to create therapeutic cell populations will increase the availability and utility of new cell therapies.

[0007] In this regard, embodiments of the invention include methods of using PSCs to generate monoclonal TCR-armed genetically engineered T (TARGET) cells, immune cells useful in a wide variety of therapeutic settings. The methods of the invention can include introducing a selected monoclonal T cell receptor (TCR) gene into the TARGET cells as an endogenous TCR gene when T cell reprogramming-induced PSC (T-iPSC) lines are used to generate the TARGET cells, or introducing a selected monoclonal TCR gene into the TARGET cells as an exogenous TCR transgene when non-T-iPSC PSC lines are used to generate the TARGET cells. The TCR transgene can include, but is not limited to, a nucleic acid molecule encoding a TCR selected from an αβ TCR (conventional CD4αβ TCR, conventional CD8αβ TCR, or non-conventional αβ TCR); a γδ TCR (Vγ9V52 TCR, V51 TCR, or other γδ TCR); an invariant NKT TCR (iNKT TCR); a non-invariant NKT TCR; and / or a mucosal-associated invariant TCR (MAIT TCR). Embodiments of the present invention include monoclonal TCR-armed genetically engineered T (TARGET) cells produced by the methods disclosed herein.

[0008] In certain embodiments of the invention, the TARGET cells comprise a gene expression profile characterized by at least one monoclonal TCR positive CD3 positive; HLA-I low / negative; HLA-II low / negative; expression of an immunomodulatory transgene and / or a suicide / marker transgene; and / or disrupted expression of endogenous immunomodulatory genes. In certain embodiments of the invention, the transgene delivered to the TARGET cells can encode any of the following: immune targeting molecules (e.g., chimeric antigen receptors, CARs; T cell receptors, TCRs; natural or synthetic receptors / ligands, and others), immune regulatory molecules (e.g., IL-2, IL-4, IL-7, IL-10, IL-12, IL-15, IL-18, IL-21, IFN-γ, TNF-α, TL1A, CD27, CD28, 4-1BB, OX40, ICOS, DAP10, Bcl11b, Batf3, ThPOK, FOXP3, Runx3, and others), immune allorejection resistance molecules (e.g., HLA-C, HLA-E, HLA-G, CD47, and others), and / or suicide control and imaging marker molecules (e.g., sr39TK, iCasp9, CD20, and others). In certain embodiments of the present invention, the endogenous genes disrupted in the TARGET cells can encode any of immune checkpoint molecules (e.g., PD-1, PD-L1, CTLA-4, LAG-3, TIM-3, TIGIT, B7-H3 / B7-H4, BTLA, VISTA, NKG2A, A2aR, PVRIG, IDO, CD73, CD39, CD96, CD161, and others), immune regulatory molecules (e.g., TET2, PI3Kδ / γ, DGK, DNMT3a, Suv39h1, and others), and / or immune allorejection molecules (e.g., HLA-I / II, B2M, CIITA, and others).

[0009] In the methods of the present invention, a single transgene or multiple transgenes can be integrated into a TARGET cell product via any of a variety of gene delivery vectors (e.g., lentivectors, retrovectors, adenovectors, AAV, and others) and / or vector-free systems (e.g., CRISPR, TALEN, Zinc-Finger, and others), and a single endogenous gene or multiple endogenous genes of the TARGET cell product can be disrupted from expression via any of a variety of gene editing tools (e.g., CRISPR, TALEN, Zinc-Finger, and others). In some embodiments of the present invention, an all-in-one engineering (AO-Engineering) strategy can be used where all the desired genetic modifications intended for a designated TARGET cell product are integrated into a master PSC line. In some embodiments of the present invention, an assembly-line engineering (AL-Engineering) strategy can be employed where all the desired genetic modifications intended for a designated TARGET cell product are generated stepwise on a master PSC line and its progeny hematopoietic stem and progenitor cells (HSPCs or HSCs; both terms are used interchangeably in the present document).

[0010] In the methods of the present invention, PSCs are cultured ex vivo to produce TARGET cells. In certain embodiments of the present invention, the ex vivo culture can be divided into three to four stages: Stage 0 (generation and maintenance of a PSC master cell bank), Stage 1 (ex vivo PSC-HSPC differentiation), Stage 2 (ex vivo HSPC-TARGET cell differentiation), and Stage 3 (ex vivo TARGET cell expansion). In certain embodiments of the present invention, an additional "CD4 induction step" can be added between Stage 2 and Stage 3 cultures to allow for the generation of CD4+ TARGET cells, and another additional "T H A "polarization step" is further added to stage 3 cultures to induce T HThis allows for the generation of polarized CD4+ TARGET cells. In certain embodiments of the present invention, all three culture stages can be feeder-free and / or serum-free, while in other embodiments of the present invention, the stage 3 culture can contain feeder cells (e.g., artificial antigen-presenting cells; APCs). In certain embodiments of the present invention, the cell culture medium can comprise a basal medium supplemented with one or more factors selected to promote the differentiation, proliferation, and sublineage commitment of PSC-derived TARGET cells (see the Detailed Description of the Invention section). In certain embodiments of the present invention, all three stages of ex vivo culture (stages 1, 2, and 3) can achieve high purity, eliminating the need for in-process purification steps. In certain embodiments of the present invention, PSC-derived HSPCs and / or HSPC-derived TARGET intermediate cell products can be freshly cultured or cryopreserved and then thawed for continued culture. In certain embodiments of the present invention, all-in-one engineered (AO-Engineered) master PSC lines are cultured ex vivo to produce a specified TARGET cell product without the need for additional genetic engineering steps. In other embodiments, Assembly-Line Engineered (AL-Engineered) master PSC lines are cultured ex vivo to generate specified TARGET cell products, requiring additional genetic engineering steps on PSC-derived HSPCs and / or other TARGET cell precursors.

[0011] In some embodiments, the TARGET cell product produced by the methods described herein can be cryopreserved. In some embodiments, the cryo-harvested cell product can be stable at room temperature for at least 1 hour. In some embodiments, the cryo-harvested cell product is stable at room temperature for at least 1, 2, 3, 4, 5, 6, 7, 8, 10, 15, 20, 24, 30, or 48 hours (or any derivable range therein). In certain embodiments, the cell product contains a solution comprising one or more of dextrose, electrolytes, albumin, dextran, and / or DMSO. In further embodiments, the cell product is in a solution that is sterile, nonpyogenic, and isotonic.

[0012] Methods of treating a patient with a TARGET cell product are also provided. In certain embodiments, the patient has cancer. In other embodiments, the patient has a viral, bacterial, fungal, or parasitic infection. In some embodiments, the patient has a disease or condition involving inflammation, excluding cancer in some embodiments. In certain embodiments, the patient has an autoimmune disease or condition. In some embodiments, the TARGET cell product is allogeneic with respect to the patient. In further embodiments, the patient does not show signs of rejection or depletion of the TARGET cells. Some treatment methods further comprise administering to the patient a stimulatory reagent that activates the TARGET cells or a reagent that triggers a kill switch of a suicide gene.

[0013] The foregoing has outlined rather broadly the features and technical advantages of the present disclosure in order that the detailed description that follows may be better understood. Additional features and advantages will be described hereinafter which form the subject of the claims herein. It should be understood by those skilled in the art that the conception and specific embodiments disclosed may be readily utilized as a basis for modifying or designing other structures for carrying out the same purposes of the present design. It should also be understood by those skilled in the art that such equivalent constructions do not depart from the spirit and scope as set forth in the appended claims. The novel features believed characteristic of the designs disclosed herein, both as to organization and method of operation, together with further objects and advantages, will be better understood from the following description when considered in connection with the accompanying drawings. It is to be expressly understood, however, that each of the figures is provided for the purpose of illustration and description only and is not intended as a definition of the limits of the present disclosure. BRIEF DESCRIPTION OF THE DRAWINGS For a more complete understanding of the present disclosure, reference is now made to the following descriptions taken in conjunction with the accompanying drawings, in which: [Brief explanation of the drawings]

[0014] [Figure 1]Overview of the PSC-T Invention. Schematic diagram showing the generation of an "off-the-shelf" PSC-derived T cell product from pluripotent stem cells (PSCs). (a) PSCs can be derived from commercially available or proprietary (in-house) sources. These sources include embryonic stem cells (ESCs), induced pluripotent stem cells (iPSCs) derived from CD34+ hematopoietic stem and progenitor cells (HSPC-iPSCs or HSC-iPSCs), iPSCs derived from mature T cells (T-iPSCs), and iPSCs derived from other non-T hematopoietic or non-hematopoietic cells (non-T iPSCs). Examples of T-iPSCs include iPSCs derived from conventional alpha-beta T cells (Tc-iPSCs), iPSCs derived from mucosal-associated invariant T cells (MAIT-iPSCs), iPSCs derived from natural killer T cells (NKT-iPSCs), and iPSCs derived from gamma-delta T cells (γδT-iPSCs). (b) Genetic engineering strategies and toolboxes include: (b1) "All-in-One" Engineering (AO-Engineering) strategy: All genetic engineering operations are performed at the PSC stage, generating a designated PSC master cell line that can be banked and used to produce a specified T cell product via streamlined PSC-T differentiation culture without the need for further genetic engineering. (b2) "Assembly-Line" Engineering (AL-Engineering) strategy: Genetic engineering operations can be performed stepwise at the PSC stage and the PSC-derived CD34+ HSPC stage, allowing for flexible "plug-in" of various genetic operations to generate an array of specified T cell products. (b3) Genetic engineering toolboxes include genetic engineering methods such as viral and non-viral vectors (e.g., lentivectors) and gene editing tools (e.g., CRISPR KO / KI), as well as genes of interest (e.g., TCR, CAR, immunomodulatory genes). (c) Scalable ex vivo PSC-T cell culture method: a chemically defined, feeder-free and serum-free culture method that can differentiate selected PSCs into mature T cells and is compatible with any kind of PSC source and selected engineering strategy.(d) "Off-The-Shelf" PSC-derived T cell products are included: the end products are various types of PSC-derived T (PSCT) cells, such as conventional T (PSCTc), natural killer T (PSCNKT), mucosal-associated invariant T (PSCMAIT), gamma-delta T (PSCγδT), and other PSCT cells, characterized by high yield, high purity, robustness, and monoclonal TCR suitable for therapeutic applications. [Figure 2]Generation and characterization of MAIT-reprogrammed iPSC (MAIT-iPSC) lines. (A) Schematic diagram showing the experimental design for generating MAIT-iPSC lines. MAIT cells were isolated from healthy donor peripheral blood mononuclear cells (PBMCs) by MR1-5-OP-RU tetramer staining followed by magnetic-activated cell sorting (MACS). Isolated PBMC-derived MAIT (PBMC-MAIT) cells were enriched from approximately 5% to over 90%. The isolated PBMC-MAIT cells were then subjected to reprogramming using the CytoTune™-iPS 2.0 Sendai Reprogramming Kit (ThermoFisher Scientific, A16517). After successful reprogramming, individual MAIT-iPSC clones were selected for further validation and characterization, including assessment of iPSC pluripotency, confirmation of MAIT TCR rearrangement, evaluation of trilineage differentiation potential (ectoderm, mesoderm, and endoderm), Sendai virus vector elimination, mycoplasma testing, and karyotype analysis. (B) Fluorescence-activated cell sorting (FACS) plot showing enrichment of PBMC-MAIT cells (gated as CD3+Vα7.2+) after MR1-5-OP-RU tetramer-mediated MACS sorting. (C) Microscopic image showing the morphology of a representative MAIT-iPSC line. Three previously established PSC lines were included as controls: H1 ESC (WiCell, WA01) is a human embryonic stem cell line, iPS21 (ALSTEM) is a human fibroblast-reprogrammed iPSC line, and T-iPSC (UCLA) is a human T cell-reprogrammed iPSC line. (D) FACS plot showing pluripotency markers (i.e., EpCAM, SSEA-4, TRA-1-81, and TRA-1-60) expressed in a representative MAIT-iPSC line. (E) DNA gel image showing the detection of recombined MAIT TCR (Vα7.2 chain) PCR products from nine reprogrammed MAIT-iPSC clones. L: DNA ladder; Samples #1 and #2: PBMC-MAIT cells as a positive control; Sample #3: H1 ESC as a negative control; Samples #4–#12: MAIT-iPSC single clones. The expected PCR product size is 312 bp. [Figure 3]Differentiation of PSCs into CD34+ HSPCs in scalable feeder-free / serum-free ex vivo culture (without genetic manipulation). (A) Schematic showing the generation of PSC-derived T cells in scalable feeder-free / serum-free ex vivo culture, highlighting the initial steps for differentiating PSCs into CD34+ HSPCs. PSCs tested include H1 ESCs (WiCell, WA01; human embryonic stem cell line), iPS21 (ALSTEM; human fibroblast-reprogrammed iPSC line), T-iPSCs (UCLA; human T cell-reprogrammed iPSC line), and MAIT-iPSCs (in-house; human MAIT cell-reprogrammed iPSC line). (B) Microscopic images showing the formation of embryoid bodies (EBs; left) on day 5 of ex vivo HSPC differentiation culture and the emergence of hematopoietic stem and progenitor cells (HSPCs; right) on day 13. Data generated using H1 ESCs are presented. (C) FACS plot showing the detection of HSPC markers (i.e., CD34, CD31, CD43, CD44, CD45, CD144, and CD235a) on cells differentiated from PSCs for 13 days. Data generated using H1 ESCs is presented. The goal of HSPC differentiation is to obtain CD34+ HSPCs that have undergone defined hematopoiesis, signifying enhanced hematopoietic commitment and lymphoid lineage potential. Throughout the differentiation process from PSCs, hemogenic endothelial cells (HECs) initially express CD34 and CD31, followed by upregulation of CD43 upon commitment to a hematopoietic fate. CD44 serves as a marker of endothelial-hematopoietic transition (EHT), which is crucial for generating adult HSPCs from PSCs. CD144 indicates endothelial cells, while CD235a, an erythroid marker, indicates primitive hematopoiesis. Furthermore, CD45 is utilized to confirm the identity of our HSPCs, as it is a hematopoietic lineage-restricted antigen expressed on all hematopoietic cells. (D) Quantification of (C) showing the percentage (left) and yield (right) of CD34+CD45+ cells at day 13 of ex vivo HSPC differentiation culture (n = 3; n indicates biological repeats). The source of starting PSCs is indicated. Data are presented as mean ± SEM. [Figure 4]Differentiation of PSC-derived CD34+ HSPCs into T cells in scalable ex vivo feeder-free / serum-free culture (without genetic manipulation). (A) Schematic showing the generation of PSC-derived T cells in scalable feeder-free / serum-free ex vivo culture, highlighting the second step for differentiating PSC-derived CD34+ HSPCs into T cells. Note that the resulting T cells are expected to express a pair of successfully rearranged endogenous TCRs, either generated during the differentiation culture if non-T-iPSCs are used, or inherited from a pre-rearranged endogenous TCR if T-iPSCs are used. (B) FACS analysis of T cell differentiation in ex vivo T cell differentiation culture over a 5-week period. Data are presented for H1 ESC-derived CD34+ HSPCs. Note that both alpha-beta T cells and gamma-delta T cells are generated. [Figure 5]Characterization of PSC-derived T cells (without genetic manipulation). (A) Schematic diagram showing the generation of PSC-derived T cells in scalable feeder-free / serum-free ex vivo culture, highlighting the characterization of the resulting PSCT cell products. Three cell products were characterized: the H1 ESC-derived conventional T cell (PSCTc) product, the T-iPSC-derived conventional T cell (PSCTc) product, and the MAIT-iPSC-derived MAIT cell (PSCMAIT) product. (B and C) PSC-derived T cell products were stimulated with 1 μg / ml anti-CD3 / CD28 (for PSCTc) or 1 μM MAIT-activating ligand, 5-(2-oxopropylideneamino)-6-D-ribitylaminouracil (5-OP-RU; for PSCMAIT) in the presence of irradiated healthy donor PBMCs as antigen-presenting cells (APCs) for 14 days, after which antigen responses of these PSC-derived T cell products were analyzed. (B) FACS plot showing expression of TCR (conventional αβ TCR or MAIT TCR) and co-receptor (primarily CD4-CD8αβ+) on the indicated PSCT cell products. (C) Quantification of antigen-stimulated proliferation of the indicated PSCT cell products (n = 3–5; n indicates biological repeats). All PSCT cell products tested showed significant proliferation in response to TCR antigen stimulation, supporting the functionality of these PSC-derived T cells. Data are presented as mean ± SEM. **P<0.01, ****P<0.0001 by Student's t-test. [Figure 6]Generation of PSC-derived MAIT cells and their CAR / IL-15-armed derivatives (AO-Engineering strategy). (A) Schematic showing the experimental design for generating PSC-derived MAIT cells and their CAR / IL-15-armed derivatives using the "All-in-One" Engineering (AO-Engineering) strategy. A selected PSC line is engineered with a designated lentivector delivering all genes of interest (MAIT TCR gene with / without additional CAR and IL-15 genes) to establish a master engineered PSC line, followed by a streamlined three-stage ex vivo culture to generate the designated PSC-derived MAIT cell product. Master PSC lines engineered with the Lenti / MAIT vector encoding the human MAIT TCR gene can give rise to conventional MAIT cells (termed PSCMAIT cells). Master PSC lines engineered with the Lenti / MAIT-BCAR vector encoding the human MAIT TCR gene together with the BCAR gene can give rise to BCAR-armed MAIT cells (termed PSCCBCAR-MAIT cells). Meanwhile, master PSC lines engineered with the Lenti / MAIT-BCAR-IL15 vector encoding the human MAIT TCR gene together with the BCAR gene and IL-15 gene can give rise to BCAR / IL-15-armed MAIT cells (termed PSC15BCAR-MAIT cells). BCAR, BCMA-targeting chimeric antigen receptor. (B) FACS monitoring of PSCMAIT cell development over time. H1 ESC lines engineered with the Lenti / MAIT vector were used to initiate ex vivo culture. (C) FACS monitoring of PSC15BCAR-MAIT cell development over time. H1 ESC lines engineered with the Lenti / MAIT-BCAR-IL15 vector were used to initiate ex vivo culture. (D) Estimated yields of H1 ESC-derived PSCMAIT, PSCCBCAR-MAIT, and PSC15BCAR-MAIT cell products based on fold expansion (n = 5; n indicates biological replicates). Data are presented as mean ± SEM. Note the robust and high yields of all three cell products. [Figure 7]Characterization of PSC-derived MAIT cells and their CAR / IL-15-armed derivatives (AO-Engineering strategy). PSCMAIT and PSC15BCAR-MAIT cell products derived from H1 ESCs via the AO-Engineering strategy were studied for their in vitro antitumor efficacy and mechanism of action (MOA). Killing of the human ovarian cancer cell line OVCAR3-FG (BCMA-) and the human multiple myeloma cell line MM.1S-FG (BCMA+) was investigated. (A and B) In vitro killing of OVCAR3-FG and MM.1S-FG tumor cells by PSCMAIT cells. (A) Experimental design. (B) Tumor cell killing data collected after 24 hours of coculture with or without the addition of 5-OP-RU. Healthy donor PBMC-derived T (denoted T) cells were included as a control (n = 3). (C and D) In ​​vitro killing of MM.1S-FG tumor cells by PSCMAIT and PSC15BCAR-MAIT cells. (C) Experimental design. (D) Tumor cell killing data collected 24 hours after coculture. Unmanipulated and BCAR-manipulated T cells (denoted as T cells and BCAR-T cells, respectively) from healthy donor PBMCs were included as controls (n = 3). (E and F) In vitro killing of OVCAR3-FG and MM.1S-FG by PSCMAIT cells with or without the addition of NKG2D / DNAM-1 blocking antibody (10 μg / ml). (E) Experimental design. (F) OVCAR3-FG (E:T ratio = 0.5:1; n = 3) and MM.1S-FG (E:T ratio = 5:1; n = 3) killing data collected 24 hours after coculture. Data are presented as mean ± SEM. ns, not significant; *P<0.05, ***P<0.001, ****P<0.0001 by one-way ANOVA (F). [Figure 8]Generation of diverse PSC-derived CAR / IL-15-armed MAIT and conventional T cell products (AL-Engineering strategy). (A) Schematic diagram showing the experimental design for generating diverse PSC-derived T cell products using various PSC sources and the "Assembly-Line" engineering (AL-Engineering) strategy. Genetic manipulation can be performed at both the PSC and CD34+ HSPC stages. Examples shown include the generation of H1 ESC-derived PSC15BCAR-MAIT cells, iPS21-derived PSC15BCAR-MAIT cells, MAIT-iPSC-derived PSC15BCAR-MAIT cells, and T-iPSC-derived PSC15BCAR-Tc cells. Note that in these examples, a single lentivector was used to deliver all transgenes at the CD34+ HSPC stage. PSC-derived CD34+ HSPCs were pre-generated, cryopreserved, and then thawed for lentivector transduction, allowing for continued culture. (B) FACS plots showing the characteristics of the indicated PSC cell products. (C) Estimated yield of the indicated PSCT cell products based on fold expansion (n=5; n indicates biological repeats). Data are presented as mean ± SEM. [Figure 9] Pharmacology Study—PSC15BCAR-MAIT Cells (AL-Engineering Strategy). PSC15BCAR-MAIT cells derived from H1 ESCs using the AL-Engineering strategy (shown in Figure 8A) were studied. Conventional T cells and MAIT cells derived from healthy donor PBMCs, as well as conventional T cells engineered to express the same BCMA-targeted CAR (denoted as PBMC-T, BCAR-T, and PBMC-MAIT cells, respectively), were included as controls. Representative FACS plots are presented. Note that PSC15BCAR-MAIT cells exhibit a phenotype similar to that of PBMC-MAIT cells (e.g., expression of high levels of effector molecules such as CD28 costimulatory molecules, mixed T / NK markers, and cytokines and cytotoxic molecules) and possess enhanced memory characteristics (e.g., enhanced expression of memory markers such as CD45RA and CD62L). [Figure 10]In vitro antitumor efficacy and mechanism of action (MOA) study of PSC15BCAR-MAIT cells (AL-Engineering strategy). PSC15BCAR-MAIT cells, derived from H1 ESCs using the AL-Engineering strategy (shown in Figure 8A), were studied. Conventional T cells derived from healthy donor PBMCs, either engineered or not, to express the same BCMA-targeted CAR (denoted as T cells and BCAR-T cells, respectively), were included as controls. The tumor cell lines used in this study include MM.1S-FG (BCMA+), a human multiple myeloma (MM) cell line engineered to express a firefly luciferase and green fluorescent protein (FG) dual reporter; BCMA-KOMM.1S-FG (BCMA-), an MM.1S-FG tumor cell line in which the BCMA gene was knocked out via CRISPR; and K562-FG, a human chronic myeloid leukemia (CML) cell line engineered to express a FG dual reporter. (A and B) Study of the in vitro antitumor efficacy of PSC15BCAR-MAIT cells against MM.1S-FG tumor cells, along with other PSC-derived T cell products generated using the AL-Engineering strategy, including iPSC-derived PSC15BCAR-MAIT, T-iPSC-derived PSC15BCAR-Tc, and MAIT-iPSC-derived PSC15BCAR-MAIT cells (shown in Figure 19A). (A) Experimental design. (B) Tumor cell killing data at 24 h (E:T ratio = 0.5:1; n = 4). Note that all PSC-derived BCAR-armed T cell products, despite their diverse PSC sources, exhibit potent antitumor efficacy comparable to that of conventional BCAR-T cells. (C and D) Testing the in vitro antitumor efficacy of PSC15BCAR-MAIT cells under repeated tumor challenge, MM.1S-FG tumor cell studies. (C) Experimental design. PSC15BCAR-MAIT cells were mixed with MM.1S-FG (E:T ratio = 1:1) and rechallenged every 2 days. Tumor cell killing data were collected on the day of rechallenge. (D) Tumor cell killing data (n = 4). Note that PSC15BCAR-MAIT cells exhibit potent and durable antitumor effects comparable to those of conventional BCAR-T cells.(E and F) Study of the tumor targeting mechanism of PSC15BCAR-MAIT cells mediated by TCR and CAR. (E) Experimental design. 5-OP-RU is the agonist antigen recognized by the MAIT TCR. (F) Tumor cell killing data at 24 hours (n = 3). (G and H) Study of the tumor targeting mechanism of PSC15BCAR-NKT cells mediated by NKR (i.e., NKG2D and DNAM-1). (G) Experimental design. (H) Tumor cell killing data at 24 hours (E:T ratio = 5:1 for BCMA-KOMM.1S-FG and 2:1 for K562-FG; n = 4). (I) Schematic diagram showing the CAR / TCR / NKR triple targeting mechanism utilized by PSC15BCAR-MAIT cells to attack BCMA+ tumor cells. Data are presented as mean ± SEM. ns, not significant; *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001 by one-way ANOVA (A,H). [Figure 11] In vivo antitumor efficacy study—PSC15BCAR-MAIT cells (AL-Engineering strategy). PSC15BCAR-MAIT cells derived from E1 HSCs using the AL-Engineering strategy (shown in Figure 8A) were studied. Conventional T cells derived from healthy donor PBMCs, engineered to express the same BCMA-targeted CAR (denoted as BCAR-T cells), were included as a benchmark control. (A) Experimental design. The MM.1S-FG human MM xenograft NSG mouse model was used. BLI: live animal bioluminescence imaging. (B) BLI images showing the presence of tumor cells in experimental mice over time. (C and D) Quantification of B, shown as the average tumor burden in all experimental mice in each treatment group over time (C) or as the tumor burden in individual experimental mice in each treatment group (D). N=5. TBL, whole body luminescence. Data are presented as mean ± SEM. ****P<0.0001 by one-way ANOVA (C). Note that PSC15BCAR-MAIT cells exhibit potent in vivo antitumor effects comparable to those of conventional BCAR-T cells. [Figure 12]Safety and immunogenicity study—PSC15BCAR-MAIT cells (AL-Engineering strategy). PSC15BCAR-MAIT cells derived from E1 HSCs using the AL-Engineering strategy (shown in Figure 19A) were studied. Conventional T cells derived from healthy donor PBMCs engineered to express the same BCMA-targeting CAR (denoted as BCAR-T cells) were included as a benchmark control. (A and B) Study of graft-versus-host (GvH) responses of PSC15BCAR-MAIT cells using an in vitro mixed lymphocyte reaction (MLR) assay. Irradiated PBMCs from randomly mismatched healthy donors were used as stimulator cells. Data from three representative donors are presented. (A) Experimental design. (B) ELISA analysis of IFN-γ production on day 4 (n=3). N, no stimulatory PBMCs added. (C-E) Study of host-versus-graft (HvG) responses of PSC15BCAR-MAIT cells using an in vitro mixed lymphocyte reaction (MLR) assay. PBMCs from random mismatched healthy donors were used as responder cells. Data from one of three representative donors are presented. (C) Experimental design. (D) ELISA analysis of IFN-γ production on day 4 (n=3). None, no addition of stimulatory treatment cells. (E) FACS measurement of HLA-I and HLA-II expression on the indicated treatment cells. MFI, mean fluorescence intensity. Data are presented as mean ± SEM. ns, not significant; *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001 by one-way ANOVA (B,D). Note that PSC15BCAR-MAIT cells did not induce GvH responses, in clear contrast to control conventional BCAR-T cells, likely due to the monoclonal MAIT TCR expressed on these PSC15BCAR-MAIT cells, which recognizes non-polymorphic MHC-I molecules such as those called MR1. It should also be noted that PSC15BCAR-MAIT cells elicited a significantly reduced HvG response compared with that elicited by conventional BCAR-T cells, likely due to their significant surface expression of HLA-I / II molecules.These "low GvHD risk" and "low immunogenicity" characteristics may confer safety of PSC15BCAR-MAIT cells and resistance to allogeneic rejection by host T cells, and thus may be attractive for allogeneic "off-the-shelf" applications of these PSC15BCAR-MAIT cells. [Figure 13] Study of Tumor Microenvironment (TME) Targeting - PSC15BCAR-MAIT Cells (AL-Engineering Strategy). E1 HSC-derived PSC15BCAR-MAIT cells were studied using the AL-Engineering strategy (shown in Figure 8A). Primary bone marrow (BM) samples collected from multiple myeloma (MM) patients were used as tumor samples in the study. (A) Experimental design for studying TME targeting by therapeutic cells. Primary BM samples collected from MM patients were cocultured with therapeutic cells (i.e., PSC15BCAR-MAIT cells) for 24 hours. (B) FACS measurement of surface MR1 expression on the indicated TME component cells in MM patient BM samples. TAM, tumor-associated macrophages; MDSC, myeloid-derived suppressor cells; T, T cells; B, B cells; NK, natural killer cells. MFI, mean fluorescence intensity. (C) Killing of the indicated TME component cells by PSC15BCAR-MAIT cells at 24 hours (n = 4; n indicates a different MM patient BM sample). Data are presented as mean ± SEM. ns, not significant; ****P < 0.0001 by Student's t-test (C). Note that PSC15BCAR-MAIT cells effectively and selectively depleted immunosuppressive TAMs and MDSCs while sparing other immune cells in the TME of primary MM patient BM samples, likely due to the high surface expression of MR1 on these TAMs and MDSCs, which are recognized by the MAIT TCR. The immunosuppressive TME, primarily mediated by TAMs and MDSCs, is considered a significant obstacle limiting cancer immunotherapy. The ability of PSC-engineered CAR-MAIT cells to target and modify the immunosuppressive TME is fascinating and offers these cells a unique opportunity for cancer therapeutic applications, particularly in solid tumors. [Figure 14]Generation and characterization of CD4 single-positive (CD4 SP) and CD8 single-positive (CD8 SP) PSC15BCAR-MAIT cells (AL-Engineering strategy). (A) Schematic showing the experimental design for generating H1 ESC-derived PSC15BCAR-MAIT cells that are either CD4 single-positive (CD4 SP) or CD8 single-positive (CD8-SP) using the AL-Engineering strategy. Note the addition of a "CD4 induction" step between stage 2 (ex vivo T cell differentiation) and stage 3 (ex vivo T cell expansion) cultures to determine CD4 SP vs. CD8 SP PSC15BCAR-MAIT cell product. (B) FACS monitoring of the transition of PSC15BCAR-MAIT cells from CD4 / CD8 double-positive (DP) to CD4 SP after inducer treatment at week 4, or their retention as CD4 / CD8 DP in the absence of inducer treatment through week 5 of T cell differentiation. Week 4 cell cultures were treated with CD4 inducers for 16 hours, then returned to fresh medium without CD4 inducers and cultured for an additional 6 days. Note that the CD4 SP lineage-commitment transcription factor ThPOK was detected in CD4 SP PSC15BCAR-MAIT cells. (C) FACS plots showing the characteristics of the final CD4 SP and CD8 SP PSC15BCAR-MAIT cell products. (D) Estimated yields of CD4 SP and CD8 SP PSC15BCAR-MAIT cell products based on fold expansion (n = 3; n indicates biological repeats). [Figure 15]Generation and Characterization of Th0 / Th1-Like and Th2-Like CD4 SP PSC15BCAR-MAIT Cells (AL-Engineering Strategy). (A) Schematic showing the experimental design for generating H1 ESC-derived CD4 SP PSC15BCAR-MAIT cells that are Th0 / Th1-like or Th2-like using the AL-Engineering strategy. Note that a "CD4 induction" step is added between stage 2 (ex vivo T cell differentiation) and stage 3 (ex vivo T cell expansion) to determine CD4 SP PSC15BCAR-MAIT lineage commitment, and an optional "Th2 polarization" step is added at stage 3 to determine the Th0 / Th1-like or Th2-like function of the final CD4 SP PSC15BCAR-MAIT cell product. Stage 3 CD4 SP PSC15BCAR-MAIT cells were stimulated with 1 μg / ml anti-CD3 / CD28 antibody for 7 days in the presence of human IL-2 and irradiated healthy donor PBMCs as antigen-presenting cells, resulting in a Th0 / Th1-like CD4 SP PSC15BCAR-MAIT final cell product. For Th2 polarization, stage 3 CD4 SP PSC15BCAR-MAIT cells were stimulated under the same conditions but with the addition of ImmunoCult Human Th2 Differentiation Supplement (StemCell Technologies) containing human IL-4 and anti-human IFN-γ, resulting in a Th2-like CD4 SP PSC15BCAR-MAIT final cell product. (B) FACS analysis of intracellular production of cytokines and effector molecules by the indicated cells. Various H1 ESC-derived PSC15BCAR-MAIT cell products were analyzed, including those induced into CD8 SP, Th0 / Th1-like CD4 SP, and Th2-like CD4 SP. Healthy donor PBMC-derived CD8 SP and CD4 SP conventional BCAR-T cells were included as controls. Note that Th2-polarized CD4 SP PSC15BCAR-MAIT cells exhibit typical Th2-like functions, evidenced by reduced production of Th1 cytokines such as IFN-γ and enhanced production of the Th2 cytokine IL-4, in contrast to non-Th2-polarized CD4 SP PSC15BCAR-MAIT cells (high production of IFN-γ and low production of IL-4), which exhibit typical Th0 / Th1-like functions.On the other hand, CD8 SP PSC15BCAR-MAIT cells exhibit typical cytotoxic functions similar to those of CD8 SP BCAR-T cells (high production of IFN-γ and cytotoxic molecules such as perforin and granzyme B). [Figure 16] CMC Study - Generation of PSCBCAR-iNKT Cells from iPSCs. (A) Experimental design for generating a PSC-engineered BCAR-armed invariant natural killer T (PSCBCAR-iNKT) cell product. BCMA, B cell maturation antigen; CAR, chimeric antigen receptor; BCAR, BCMA-targeted CAR. (B) Schematic diagram of the Lenti / iNKT-BCAR-(GFP) lentivector encoding the human iNKT TCR gene, BCAR gene, and optional GFP reporter gene. (C) FACS plot showing retention of pluripotency markers (i.e., TRA-1-60R and SSEA-5) on the engineered DMD iPSC master cell line. Note the co-expression of the GFP reporter. (D-E) Differentiation of the engineered iPSC master cell line into HSCs during the HSC differentiation stage. (D) Microscopic images showing cell culture over time. (E) FACS plot showing detection of HSC markers (i.e., CD34, CD43, and CD31) on cells from day 12 of culture. (F-G) Differentiation of iPSC-derived HSCs into PSCBCAR-iNKT cells during the iNKT differentiation stage. (F) FACS plot showing the generation of human iNKT cells (identified as CD3+iNKT TCR+) over time. (G) FACS plot showing co-expression of iNKT TCR, BCAR, and GFP reporter on mature PSCBCAR-iNKT cells harvested on day 28 of iNKT differentiation stage culture. [Figure 17]CMC Research - Generation of PSC BCAR-iNKT Cells from ESCs. (A) DNA gel image showing detection of the transgene in PSC master cell lines (H1 ESC line and DMD iPSC line) transduced with the lenti / iNKT-BCAR-GFP vector. Genomic DNA was extracted from the indicated PSC master cell lines and subjected to polymerase chain reaction (PCR) to amplify a partial transgene fragment of approximately 2,700 bp. (B) FACS plot showing retention of pluripotency markers (i.e., TRA-1-60R and SSEA-5) on the genetically engineered H1 ESC master cell line. Note the co-expression of the GFP reporter. (C-D) Differentiation of the genetically engineered ESC master cell line into HSCs during the HSC differentiation stage. (D) Microscopic images showing cell cultures over time. (E) FACS plot showing detection of HSC markers (i.e., CD34, CD43, and CD31) on cells from day 12 of culture. (E-F) Differentiation of ESC-derived HSCs into PSCBCAR-iNKT cells at the iNKT differentiation stage. (E) FACS plot showing the generation of human iNKT cells (identified as CD3+iNKT TCR+) over time. (F) FACS plot showing co-expression of iNKT TCR, BCAR, and GFP reporter on mature PSCBCAR-iNKT cells harvested at day 28 of iNKT differentiation stage culture. [Figure 18] Pharmacology study - PSCBCAR-iNKT cells. Representative FACS plots showing phenotypic (surface markers) and functional (intracellular production of effector molecules) analysis of PSCBCAR-iNKT cells. Data are presented for DMD iPSC-derived PSCBCAR-iNKT cells. Conventional αβ T cells derived from healthy donor PBMCs (denoted BCAR-T cells) engineered to express the same BCAR were included as a benchmark control. [Figure 19]In vitro efficacy study - PSCiNKT cells. In vitro direct killing of human tumor cells by PSCiNKT cells was studied. Conventional αβ T (PBMC-T) cells derived from healthy donor PBMCs were included as a control. Data for DMD iPSC-derived PSCiNKT cells are presented. Four human tumor cell lines were studied: A375 (melanoma), MDA (breast cancer), OVCAR8 (ovarian cancer), and PC3 (prostate cancer). All four tumor cell lines were engineered to express a firefly luciferase and green fluorescent protein (FG) dual reporter. N=4. (A) Experimental design. (B-E) Tumor cell killing data at 24 hours. FACS plots showing upregulation of surface activation markers (i.e., CD69) and intracellular cytotoxic molecules (i.e., perforin and granzyme B) in PSCiNKT cells after coculture with tumor cells, as well as ELISA analysis of cell culture supernatants showing increased IFN-γ secretion by PSCiNKT cells after coculture with tumor cells, are also presented. Data are presented as mean ± SEM. ns, not significant; *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001 by Student's t-test. [Figure 20]MOA study - PSCiNKT cells. Direct killing of human tumor cells by PSCiNKT cells via the NK pathway was studied. Data are presented for DMD iPSC-derived PSC BCAR-iNKT cells. Conventional αβ T (PBMC-T) cells derived from healthy donor PBMCs were included as a control. Four human tumor cell lines were studied: A375 (melanoma), MDA (breast cancer), OVCAR8 (ovarian cancer), and PC3 (prostate cancer). All four tumor cell lines were engineered to express firefly luciferase and green fluorescent protein (FG) dual reporters. (A) Experimental design. The NK-activating receptor DNAM-1-mediated pathway was studied. (B) Tumor cell killing data at 24 hours (tumor:effector cell ratio 1:2; n=3). Data are presented as mean ± SEM. ns, not significant; *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001 by one-way ANOVA. (C) FACS plots showing the detection of DNAM-1 ligand stress molecules (i.e., nectin-2 / CD112 and PVR / CD155) on the indicated human tumor cell lines. [Figure 21]In vitro efficacy and MOA study - PSCBCAR-iNKT cells. Data exists for DMD iPSC-derived PSCBCAR-iNKT cells. (A-B) In vitro direct killing of MM.1S-FG tumor cells. (A) Experimental design. (B) Tumor killing data at 24 hours (n=3). (C-D) In ​​vitro direct killing of MM.1S-CD1d-FG tumor cells. (C) Experimental design. (D) Tumor killing data at 24 hours (n=3). MM.1S, human multiple myeloma cell line; MM.1S-FG, MM.1S cell line engineered to express firefly luciferase and green fluorescent protein dual reporter; MM.1S-CD1d-FG, MM.1S cell line engineered to express human CD1d and firefly luciferase and green fluorescent protein dual reporter. Four effector cells were studied: conventional αβ T cells derived from healthy donor PBMCs (PBMC-T), BCMA-targeted CAR-engineered PBMC-T (BCAR-T), PSC-derived iNKT (PSCiNKT), and PSC-derived BCMA-targeted CAR-armed iNKT (PSCBCAR-iNKT) cells. Data are presented as mean ± SEM. ns, not significant; *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001 by one-way ANOVA. [Figure 22]Safety and immunogenicity study - PSCBCAR-iNKT cells. Data exists for DMD iPSC-derived PSCBCAR-iNKT cells. (A-B) In vitro mixed lymphocyte reaction (MLR) assay to study graft-versus-host (GvH) responses. PSCBCAR-iNKT cells were studied as responder cells. Conventional BCAR-T cells derived from healthy donor PBMCs were included as responder controls. Irradiated donor-mismatched PBMCs from three random healthy donors were used as stimulator cells. (A) Experimental design. (B) ELISA analysis of IFN-γ production on day 4 (n=3). N, no stimulator cells. (C-D) In ​​vitro mixed lymphocyte reaction (MLR) assay to study graft-versus-host (GvH) responses. Irradiated PSCBCAR-iNKT cells were studied as stimulator cells. Conventional BCAR-T cells derived from irradiated healthy donor PBMCs were included as stimulator controls. Donor-mismatched PBMCs from three random healthy donors were used as responder cells. (C) Experimental design. (D) ELISA analysis of IFN-γ production on day 4 (n=3). (E) FACS plot showing detection of surface HLA-I (B2M) and HLA-II molecules on PSCBCAR-iNKT cells. Conventional BCAR-T cells derived from healthy donor PBMCs were included as a control. (F) Quantification of E (n=3). Data are presented as mean ± SEM. ns, not significant; *P<0.05, **P<0.01, ****P<0.0001 by one-way analysis (B and D) or Student's t-test (F). [Figure 23]CMC Study - Generation of PSC γδ T Cells. (A) Experimental design for generating PSC γδ T cell products. (B) Schematic diagram of the Lenti / γδ T lentivector encoding the human γ9 and δ2 TCR gene pair. (C) DNA gel image showing transgene detection in an H1 ESC master cell line transduced with the Lenti / γδ T vector. Genomic DNA was extracted from the γδ TCR-engineered H1 ESC master cell line and subjected to polymerase chain reaction (PCR) to amplify the partial transgene fragment. An unengineered H1 ESC line was included as a control. Detection of HSC markers (i.e., CD34, CD43, and CD31) on cells from day 12 of culture. (D) Differentiation of the γδ TCR-engineered ESC master cell line into HSCs at the HSC differentiation stage. FACS plots showing detection of HSC markers (i.e., CD34, CD43, and CD31) on cells from day 12 of culture are presented. (E-F) Differentiation of ESC-derived HSCs into PSCγδT cells at the γδT differentiation stage. (E) FACS plot showing the generation of human γδT cells (identified as CD3+Vδ2 TCR+) over time. (F) FACS plot showing detection of CD16 expression on mature PSCγδT cells collected on day 28 of the γδT differentiation stage culture. [Figure 24] Pharmacology study - PSC γδ T cells. Data for H1 ESC-derived PSC γδ T cells are presented. Representative FACS plots showing analysis of the phenotype (surface markers) and functionality (intracellular production of effector molecules) of PSC γδ T cells are shown. Conventional γδ T (PBMC-T) and γδ T (PBMC-γδT) cells derived from healthy donor PBMCs were included as staining controls. [Figure 25]In vitro efficacy and MOA study - PSCγδT cells. Data for H1 ESC-derived PSCγδT cells are presented. (A-B) In vitro direct killing of A375-FG tumor cells. (A) Experimental design. (B) Tumor killing data at 24 hours (n=3). (C-D) In ​​vitro direct killing of MM.1S-FG tumor cells. (C) Experimental design. (D) Tumor killing data at 24 hours (n=3). ZOL: zoledronate, a Vγ9Vγδ2 TCR stimulator. A375-FG, a human melanoma cell line A375 engineered to express a firefly luciferase and green fluorescent protein dual reporter; MM.1S-FG, a human multiple myeloma cell line MM.1S engineered to express a firefly luciferase and green fluorescent protein dual reporter. PSC-derived human γδT (PSCγδT) cells were studied. Conventional γδ T cells derived from healthy donor PBMCs (PBMC-T) were included as a control. Data are presented as mean ± SEM. ns, not significant; *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001 by Student's t-test (B) or one-way (D). [Figure 26]Safety and immunogenicity study - PSC γδ T cells. Data for H1 ESC-derived PSC γδ T cells are presented. (A-B) In vitro mixed lymphocyte reaction (MLR) assays to study graft-versus-host (GvH) responses. PSC γδ T cells were used as responder cells. Conventional γδ T (PBMC-T) and γδ T (PBMC-γδT) cells derived from healthy donor PBMCs were included as responder controls. Irradiated donor-mismatched PBMCs from three random healthy donors were used as stimulator cells. (A) Experimental design. (B) ELISA analysis of IFN-γ production on day 4 (n=3). N, no stimulator cells. (C) FACS measurement of surface HLA-I / II molecules on the indicated cells (n=3). (D-E) In vitro mixed lymphocyte reaction (MLR) assays to study host-versus-graft (HvG) responses. Irradiated PSCγδT cells were studied as stimulator cells. PBMC-T and PBMC-γδT cells from irradiated healthy donors were included as stimulation controls. Donor-mismatched PBMCs from random healthy donors were used as responder cells. (D) Experimental design. (E) ELISA analysis of IFN-γ production on day 4 (n=3). Data are presented as mean ± SEM. ns, not significant; *P<0.05, **P<0.01, ****P<0.0001 by one-way analysis (B,C,E). DETAILED DESCRIPTION OF THE INVENTION

[0015] Detailed Description Unless otherwise defined, all technical terms, notations, and other scientific or technical terms used herein are intended to have the meaning commonly understood by those skilled in the art to which this invention pertains. In some cases, terms with commonly understood meanings may be defined herein for clarity and / or ease of reference, and the inclusion of such definitions herein should not necessarily be interpreted as representing a substantial difference from what is commonly understood in the art. Many of the techniques and procedures described or referenced herein are well understood and commonly employed by those skilled in the art using conventional methodology. Where appropriate, procedures involving the use of commercially available kits and reagents are generally performed according to manufacturer-defined protocols and / or parameters unless otherwise specified.

[0016] As discussed below, the present inventors have discovered that pluripotent stem cells (PSCs) can be genetically engineered and differentiated into different types of immune cells, thereby providing an unlimited resource for developing ready-to-use cell therapies. As known in the art, pluripotent stem cells include, for example, embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs). For a review of the current state of PSC-based cell therapy, see, for example, Zhou Y, Li M, Zhou K, Brown J, Tsao T, Cen X, Husman T, Baipai A, Dunn ZS, and Yang L. Engineering Induced Pluripotent Stem Cells for Cancer Immunotherapy. Cancers. 2022, 14:2266.

[0017] T cells play a central role in mediating and orchestrating the immune response to cancer. They are therefore attractive therapeutic targets for treating cancer and other diseases (e.g., Couzin-Frankel, J. 2013. Breakthrough of the year 2013. Cancer immunotherapy. Science 342:1432-1433; Lim, WA, and CH June. 2017. The Principles of Engineering Immune Cells to Treat Cancer. Cell 168:724-740; Rosenberg, SA, and NP Restifo. 2015. Adoptive cell transfer as personalized immunotherapy for human cancer. Science 348:62-68; Vivier, E., Ugolini, S., Blaise, D., Chabannon, C. & Brossay, L. Targeting natural killer cells and natural killer T cells in cancer. Nat Rev Immunol 12,239-52 (2012); Meraviglia S., Lo Presti (See E., Dieli F., Stassi G. 2015. T cell-based anticancer immunotherapy: progress and possibilities. Immunotherapy 7:949-951; and Godfrey DI, Le Nours J., Andrew DM, Uldrich AP, and Rossjohn J. 2018. Unconventional T cell targets for cancer immunotherapy. Immunity 48, March 20, 2018.) T cells recognize antigens through their surface T cell receptor (TCR) molecules (e.g., αβ TCR; aγδ TCR; invariant NK TCR, non-invariant NKT TCR, and / or mucosal-associated invariant TCR).Typically, the TCR polypeptide molecule presented by a T cell is encoded by a single TCR gene (which contains two genes encoding the two subunits of the TCR molecule; referred to in this material as the TCR gene). The TCR gene of a T cell can be generated by a random genomic V / D / J recombination process during T cell development and is therefore unique to each T cell. Based on the genomic composition of their TCR genes, T cells can be divided into two broad categories: alpha-beta T (αβ T) cells and gamma-delta T (γδ T) cells. Alpha-beta T cells can be further divided into subtypes: 1) CD4 T cells; + Helper T cells (CD4 T cells; or T H cells) and CD8 + 1) conventional αβ T cells, including cytotoxic T cells (CD8 T cells; or CTL) cells; 2) non-conventional αβ T cells, including type 1 invariant natural killer T (iNKT) cells, type 2 natural killer T (type 2 NKT) cells, mucosal-associated invariant T (MAIT) cells, etc.

[0018] Exemplary TCRs useful in embodiments of the present invention are discussed below.

[0019] Conventional αβ CD8 T (CD8 T) cells

[0020] CD8 T cells recognize protein peptide antigens presented by polymorphic major histocompatibility complex (MHC) class I molecules. CD8 T cells are potent cytotoxic cells for killing target pathogenic cells. CD8 T cells are also called cytotoxic T lymphocytes (CTLs).

[0021] Conventional αβ CD4 T (CD4 T) cells

[0022] CD4 T cells recognize protein peptide antigens presented by polymorphic MHC class II molecules. CD4 T cells are T helper (T) cells that orchestrate immune responses. HBased on their specialized functions, CD4 T cells are divided into further subtypes: T H 1. T H 2. T H 17, T FH , T H 9. T REG It can be classified as follows.

[0023] Type 1 invariant natural killer T (iNKT) cells

[0024] iNKT cells recognize glycolipid antigens presented by the nonpolymorphic, nonclassical MHC class I-like molecule CD1d. As a result, iNKT cells do not cause graft-versus-host disease (GvHD) when adoptively transferred into allogeneic recipients. The iNKT TCR contains an invariant alpha chain (Vα14-Jα18 in mice; Vα24-Jα18 in humans) and a limited selection of beta chains (predominantly Vβ8 / Vβ7 / Vβ2 in mice; predominantly Vβ11 in humans). Both mouse and human iNKT cells respond to the synthetic agonist glycolipid ligand alpha-galactosylceramide (αGC, or α-GC, or α-GalCer).

[0025] Type 2 natural killer T (NKT) cells

[0026] Type 2 NKT cells are also CD1d restricted. Type 2 NKT cells have a more diverse TCR repertoire and their antigens are less well defined.

[0027] MAIT cells Human mucosal-associated invariant T (MAIT) cells are characterized by the expression of the invariant TCR α chain Vα7.2-Jα33 / Jα20 / Jα12 paired with a restricted TCR β chain. MAIT cells recognize microbial peptides presented by the highly conserved MHC class I-like molecule MR1 and mediate enhanced immune responses by bridging the innate and adaptive immune systems. Upon activation, MAIT cells rapidly proliferate, produce a variety of cytokines and cytotoxic molecules, and induce efficient antitumor immunity. Administration of the representative MAIT cell ligand 5-OP-RU effectively activates MAIT cells and enhances their antitumor capabilities. See, e.g., Li YR, Zhou K, Wilson M, Kramer A, Zhu Y, Dawson N, and Yang L. Mucosal-associated invariant T cells for cancer immunotherapy. Mol Ther. 2022, Dec 5:S1525-0016(22)00677-3.

[0028] gamma-delta T cells The most comprehensively studied human γδ T subsets are Vδ1+ and Vδ2+ T cells. γδ T cells expressing the Vγ9 chain paired with the Vδ2 chain (Vγ9Vδ2 T cells) are the major γδ T cell population in human peripheral blood. Vγ9Vδ2 T cells respond to cells with accumulated intracellular phosphoantigens (pAg), intermediate metabolites produced by infected or transformed cells. These metabolites include isopentenyl pyrophosphate (IPP), formed by the mevalonate (MVA) pathway in tumor cells, and (E)-4-hydroxy-3-methyl-but-2-enyl pyrophosphate (HMB-PP), produced by microbial isoprenoid biosynthesis. Overproduction of IPP in cancer cells as a result of dysregulation of the MVA pathway leads to activation of Vγ9Vδ2 T cells. Stimulation of Vγ9Vδ2 T cells can be achieved using bisphosphonates, such as zoledronate (ZOL), a class of drugs that prevent or slow bone loss. The Vδ1+ subset has been found to recognize CD1 proteins, whereas recognition of lipid antigens on CD1 molecules is typically associated with NKT cells. See, e.g., Lee D, Rosenthal CJ, Penn NE, Dunn ZS, Zhou Y, and Yang L. Human γδ T cell subsets and their clinical applications for cancer immunotherapy. Cancers. 2022, 14(12):3035.

[0029] Other T cells

[0030] See, e.g., Godfrey DI, Le Nours J., Andrew DM, Uldrich AP, and Rossjohn J. Unconventional T cell targets for cancer immunotherapy. Immunity. 2018, 48(3):453.

[0031] CD4 helper T cell subsets In human peripheral blood, CD4 T cells make up approximately two-thirds of the total T cell population, and CD8 T cells make up the remaining one-third. H ) cells express the CD4 co-receptor. Upon encountering an antigen, naive T H Cells are activated, undergo differentiation, and release cytokines to promote and regulate immune responses. Depending on the cytokine environment, naive CD4 T cells H The cells are T H1 , T H2 , T H17 , T H9 , follicular helper T cells (T FH ) and regulatory T cells (T REG ) have the ability to polarize into various subsets, including. See, for example, Zhu X, Zhu J. CD4 T Helper Cell Subsets and Related Human Immunological Disorders. Int J Mol Sci. 2020. 21(21):8011. T H1 cell T H1 The cells are involved in cell-mediated immunity. They are characterized by the expression of the transcription factors T-bet and signal transducer and activator of transcription (STAT) 4, and the production of IL-2, IFN-γ, and TNF-α. T H2 cell T H2 These cells are mediators of humoral immunity. They develop into IL-4, IL-5, and IL-13-producing cells and are characterized by expression of the transcription factors GATA-3 and STAT6. T H17 cell T H17 These cells produce IL-17 and regulate tissue inflammation in host defense and chronic disease. They are characterized by expression of the transcription factor RORγ. T FH cell T FHB cells are essential for germinal center formation, affinity maturation, and development of most high-affinity antibody and memory B cells. They are characterized by secreting IL-6, IL-10, IL-12, and IL-21 and expressing the transcription factor Bcl6. T H9 cell T H9 These cells produce IL-9 and play a role in defense against helminth infections, allergic responses, autoimmunity, and tumor suppression. They are characterized by expression of the transcription factor PU.1. T REG cell T REG Suppressor T cells are a subpopulation of T cells that regulate the immune system, maintain tolerance to self-antigens, and prevent autoimmune diseases. They secrete the anti-inflammatory cytokines IL-10 and TGF-β and express the transcription factor Foxp3.

[0032] The invention disclosed herein has numerous embodiments. Embodiments of the invention include methods of generating monoclonal TCR-armed genetically engineered T (TARGET) cells, which may include (a) introducing a selected monoclonal T cell receptor (TCR) gene into the TARGET cells as an endogenous TCR gene when a T cell reprogrammed induced PSC (T-iPSC) line is used to generate the TARGET cells, or (b) introducing a selected monoclonal TCR gene into the TARGET cells as an exogenous TCR transgene when a non-T-iPSC PSC line is used to generate the TARGET cells. The TCR transgene can include, but is not limited to, a nucleic acid molecule encoding a TCR selected from an αβ TCR (conventional CD4αβ TCR, conventional CD8αβ TCR, or non-conventional αβ TCR); a γδ TCR (Vγ9Vδ2 TCR, δ1 TCR, or other γδ TCR); an invariant NKT TCR (iNKT TCR); a non-invariant NKT TCR; and / or a mucosal-associated invariant TCR (MAIT TCR). Embodiments of the present invention include monoclonal TCR-armed genetically engineered T (TARGET) cells produced by the methods disclosed herein.

[0033] In certain embodiments of the invention, the TARGET cells comprise a gene expression profile characterized by at least one monoclonal TCR positive CD3 positive; HLA-I low / negative; HLA-II low / negative; expression of an immunomodulatory transgene and / or a suicide / marker transgene; and / or disrupted expression of endogenous immunomodulatory genes. In certain embodiments of the invention, the transgene delivered to the TARGET cells can encode any of the following: immune targeting molecules (e.g., chimeric antigen receptors, CARs; T cell receptors, TCRs; natural or synthetic receptors / ligands, and others), immune regulatory molecules (e.g., IL-2, IL-4, IL-7, IL-10, IL-12, IL-15, IL-18, IL-21, IFN-γ, TNF-α, TL1A, CD27, CD28, 4-1BB, OX40, ICOS, DAP10, Bcl11b, Batf3, ThPOK, FOXP3, Runx3, and others), immune allorejection resistance molecules (e.g., HLA-C, HLA-E, HLA-G, CD47, and others), and / or suicide control and imaging marker molecules (e.g., sr39TK, iCasp9, CD20, and others). In certain embodiments of the present invention, the endogenous genes disrupted in the TARGET cells can encode any of immune checkpoint molecules (e.g., PD-1, PD-L1, CTLA-4, LAG-3, TIM-3, TIGIT, B7-H3 / B7-H4, BTLA, VISTA, NKG2A, A2aR, PVRIG, IDO, CD73, CD39, CD96, CD161, and others), immune regulatory molecules (e.g., TET2, PI3Kδ / γ, DGK, DNMT3a, Suv39h1, and others), and / or immune allorejection molecules (e.g., HLA-I / II, B2M, CIITA, and others).

[0034] In the methods of the present invention, a single transgene or multiple transgenes can be integrated into a TARGET cell product via any of a variety of gene delivery vectors (e.g., lentivectors, retrovectors, adenovectors, AAV, and others) and / or vector-free systems (e.g., CRISPR, TALEN, Zinc-Finger, and others), and a single endogenous gene or multiple endogenous genes of the TARGET cell product can be disrupted from expression via any of a variety of gene editing tools (e.g., CRISPR, TALEN, Zinc-Finger, and others). In some embodiments of the present invention, an all-in-one engineering (AO-Engineering) strategy can be used where all the desired genetic modifications intended for a designated TARGET cell product are integrated into a master PSC line. In some embodiments of the present invention, an assembly-line engineering (AL-Engineering) strategy can be employed where all the desired genetic modifications intended for a designated TARGET cell product are generated stepwise on a master PSC line and its progeny hematopoietic stem and progenitor cells (HSPCs or HSCs; both terms are used interchangeably in the present document).

[0035] PSCs refer to human pluripotent stem cells, which can be embryonic stem cells (ESCs) or induced pluripotent stem cells (iPSCs). iPSCs can be reprogrammed from T cells (resulting in T-iPSCs) or from non-T cells, such as CD34+ HSPCs (resulting in HSPC-iPSCs), and others (e.g., fibroblasts, NK cells, macrophages). Genetically engineered PSCs (and their derived HSPCs) can be used to establish master cell banks as an unlimited supply for generating intended "off-the-shelf" immune cell products via ex vivo culture.

[0036] In the methods of the present invention, PSCs are cultured ex vivo to produce TARGET cells. In certain embodiments of the present invention, the ex vivo culture can be divided into three stages: Stage 0 (generation and maintenance of a PSC master cell bank), Stage 1 (ex vivo PSC-HSPC differentiation), Stage 2 (ex vivo HSPC-TARGET cell differentiation), and Stage 3 (ex vivo TARGET cell expansion). In certain embodiments of the present invention, an additional "CD4 induction step" can be added between Stage 2 and Stage 3 cultures to allow for the generation of CD4+ TARGET cells, and another additional "T H A "polarization step" is further added to stage 3 cultures to induce T H This allows for the generation of polarized CD4+ TARGET cells. In certain embodiments of the present invention, all three culture stages can be feeder-free and / or serum-free, while in other embodiments of the present invention, the stage 3 culture can contain feeder cells (e.g., artificial antigen-presenting cells; APCs). In certain embodiments of the present invention, the cell culture medium can comprise a basal medium supplemented with one or more factors selected to promote the differentiation, proliferation, and sublineage commitment of PSC-derived TARGET cells (see the Detailed Description of the Invention section). In certain embodiments of the present invention, all three stages of ex vivo culture (stages 1, 2, and 3) can achieve high purity, eliminating the need for in-process purification steps. In certain embodiments of the present invention, PSC-derived HSPCs and / or HSPC-derived TARGET intermediate cell products can be freshly cultured or cryopreserved and then thawed for continued culture. In certain embodiments of the present invention, all-in-one engineered (AO-Engineered) master PSC lines are cultured ex vivo to produce a specified TARGET cell product without the need for additional genetic engineering steps. In other embodiments, Assembly-Line Engineered (AL-Engineered) master PSC lines are cultured ex vivo to generate specified TARGET cell products, requiring additional genetic engineering steps on PSC-derived HSPCs and / or other TARGET cell precursors.

[0037] In the methods of the present invention, Stage 0 cultures can support the generation and maintenance of PSC master cell banks. In some embodiments, selected PSC lines are cultured in an appropriate cell culture vessel (e.g., in Matrigel or on a laminin-coated plate) containing serum-free PSC culture medium for 12-72 hours, followed by a genetic manipulation step involving single or multiple transgene delivery vectors (e.g., lentivectors, retrovectors, adenovectors, AAV, and others) and / or vector-free systems (e.g., CRISPR, TALEN, Zinc-Finger, and others) and / or gene editing tools (e.g., CRISPR, TALEN, Zinc-Finger, and others), and then cultured for an additional 1-7 days. PSC culture media can include basal media (e.g., mTeSR™ Plus Medium, CTS™ Essential 8™ Medium, StemFit® AK03N Medium) and supplements such as bFGF, TGFβ, FLT3L, Noggin, Activin, Biotin, LIF, and others. After validation (and optional sorting and single cloning), validated genetically engineered PSC lines can be used to establish master cell banks that can be cryopreserved for storage and / or maintained in cell culture via passaging. In some embodiments, all genetic modifications of a PSC master line can be performed in a single genetic engineering step. Meanwhile, in other embodiments, genetic modifications can occur stepwise, resulting in intermediate PSC master lines that can be used for convenient "plug-in" engineering.

[0038] In the methods of the present invention, stage 1 cultures can support the differentiation of PSCs into CD34+ HSPCs. In some embodiments, genetically engineered PSC master cell lines generated and maintained from stage 0 cultures can be gently dissociated into single cells (e.g., via cell dissociation reagents such as Accutase, Versene, or TrypLE) and then transferred to appropriate cell culture vessels (e.g., ultra-low attachment plates or AggreWell) and cultured in serum-free HSPC differentiation medium A for 12-48 hours to form embryoid bodies (EBs) or monolayers. Fresh medium can then be added to the cell cultures until day 3-4. HSPC differentiation medium A may include basal media (e.g., APEL medium, StemPro-34 SFM, Ham's F-12 nutrient mix, Iscove's Modified Dulbecco's Medium, and others) and supplements such as GlutaMAX, non-essential amino acids, vitamin C (e.g., L-ascorbic acid, L-ascorbic acid 2-phosphate sesquimagnesium salt hydrate and other forms), monothioglycerol, transferrin, activin A, BMP-4, bFGF, VEGF, GSK3 inhibitors / WNT activators (e.g., Wnt3A, CHIR99021, AR-A014418, TWS119, LY2090314, 9-ING-41, and others) and ROCK inhibitors (e.g., Y-27632, thiazovivin, H-1152, and others). On days 3-4, remove half of the medium from the cell cultures and replace it with serum-free HSPC differentiation medium B, followed by culturing for an additional 6-10 days. Perform half-medium changes every other day using fresh HSPC differentiation medium B.HSPC differentiation medium B is a mixture of basal medium (e.g., APEL medium, StemPro-34 SFM, Ham's F-12 nutrient mix, Iscove's Modified Dulbecco's Medium, and others) and supplemented with GlutaMAX, non-essential amino acids, vitamin C (e.g., L-ascorbic acid, L-ascorbic acid 2-phosphate sesquimagnesium salt hydrate and other forms), monothioglycerol, human transferrin, heparin, serum-free supplements (e.g., recombinant human albumin, BIT 9500 serum replacement, B-27 supplement, and others), TGF-β inhibitors (e.g., SB431542, SB505124, SB525334, A83-01, and others), aryl hydrocarbon receptor inhibitors (e.g., PD98059, BAY-218, StemRegenin, and others). 1, CH-223191, etc.), BMP-4, bFGF, VEGF, SCF, TPO, Flt3 ligand, IL-3, IL-6, IL-11, IGF-1, IGF-2, EPO, SDF-1α, and other supplements. At the end of stage 1 culture, CD34 cells were obtained. + HSPCs can be harvested and advanced to new stage 2 cultures or cryopreserved for future use.

[0039] In the methods of the present invention, stage 2 cultures support PSC-derived HSPCs generated from stage 1 cultures to differentiate into mature TARGET cells over a period of 4-10 weeks in the absence of feeders. In some embodiments, freshly harvested PSC-derived HSPCs can proceed directly to stage 2 culture. In other embodiments, cryopreserved PSC-derived HSPCs can be recovered and then proceeded to stage 2 culture. In some embodiments, fresh or cryopreserved PSC-derived HSPCs can be cultured for 12-14 days in non-tissue culture-treated plates coated with TARGET culture coating (TARGETc) material (e.g., DLL-1 / 4, VCAM-1 / 5, Retronectin, and others) and TARGET Expansion (TARGETe) medium. TARGETe medium includes basal media (e.g., Iscove's Modified Dulbecco's Medium, RPMI 1640, SFEMII, αMEM, and others) and supplements, such as serum-free supplements (e.g., recombinant human albumin, BIT 9500 serum replacement, B-27 supplement, and others), insulin-transferrin-selenium, 2-mercaptoethanol, vitamin C (e.g., L-ascorbic acid, L-ascorbic acid 2-phosphate sesquimagnesium salt hydrate and other forms), human low-density lipoprotein, IL-7, SCF, TPO, IL-3, IL-6, Flt3 ligand, SDF-1α, HSC self-renewal agonists (e.g., UM171, UM729, and others), aryl hydrocarbon receptor inhibitors (e.g., PD98059, BAY-218, StemRegenin 1, CH-223191, and others), p38 MAPK inhibitors (e.g., Adezmapimod, Doramapimod, Losmapimod, SB202190, etc.) and other additives may be included. TARGETe Medium may be refreshed every 3-4 days. Cells may then be harvested, resuspended in TARGET Maturation (TARGETm) Medium, and cultured for an additional 14-28 days.TARGETm medium is a mixture of basal media (e.g., Iscove's Modified Dulbecco's Medium, RPMI 1640, SFEMII, αMEM, and others) and supplements, such as serum-free supplements (e.g., recombinant human albumin, BIT 9500 serum replacement, B-27 supplement, and others), insulin-transferrin-selenium, 2-mercaptoethanol, vitamin C (e.g., L-ascorbic acid, L-ascorbic acid 2-phosphate sesquimagnesium salt hydrate and other forms), human low-density lipoprotein, IL-7, SCF, Flt3 ligand, IL-2, IL-15, IL-21, TGF-β, SDF-1α, HSC self-renewal agonists (e.g., UM171, UM729, and others), aryl hydrocarbon receptor inhibitors (e.g., PD98059, BAY-218, StemRegenin 1, CH-223191, and others), p38 MAPK inhibitors (e.g., Adezmapimod, Doramapimod, Losmapimod, SB202190, and others) and other additives may be included. TARGETm Medium may be refreshed every 3 to 4 days. In some embodiments, if no additional genetic manipulation steps are performed in Stage 2 culture, an All-in-One Engineering (AO-Engineering) strategy may be applied. Meanwhile, in other embodiments, if additional genetic manipulation step(s) are performed in Stage 2 culture, an Assembly-Line Engineering (AL-Engineering) strategy may be applied. In some embodiments, the genetic manipulation step can be performed at the beginning of Stage 2 culture when cells are cultured in TARGETe Media. In some embodiments, the genetic manipulation step can involve interaction of cultured cells with single or multiple transgene delivery vectors (e.g., lentivectors, retrovectors, adenovectors, AAV, and others) and / or vector-free systems (e.g., CRISPR, TALEN, Zinc-Finger, and others) and / or gene editing tools (e.g., CRISPR, TALEN, Zinc-Finger, and others).The resulting mature TARGET cells generated at the end of Stage 2 culture can be harvested and advanced to a new Stage 3 culture or cryopreserved for future use. The intermediate TARGET progenitor cells generated during Stage 2 culture can also be advanced along a new Stage 2 culture or cryopreserved for future use.

[0040] In the methods of the present invention, Stage 3 cultures can support the expansion of mature TARGET cells generated from Stage 2 cultures over a period of approximately 1-4 weeks, resulting in the final TARGET cell product. In some embodiments, fresh or cryo-harvested mature TARGET cells generated from Stage 2 cultures can be stimulated with TCR cognate antigens (e.g., proteins, peptides, lipids, phosphoantigens, small molecules, and others) or nonspecific TCR stimulatory reagents (e.g., anti-CD3 / anti-CD28 antibodies or antibody-coated beads, concanavalin A, PMA / ionomycin, and others) with or without the presence of antigen-presenting cells (e.g., irradiated healthy donor PBMCs, artificial APCs, and others) and expanded in T Cell Culture Media for up to 1 month. Media may include T cell-supportive cytokines (e.g., IL-2, IL-4, IL-7, IL-12, IL-15, IL-18, IL-21, TNFα, TL-1A, SDF-1α, TGF-β, etc.), as well as Wnt activators or glycogen synthase kinase-3 (GSK-3) inhibitors (e.g., Wnt3A, etc.), CHIR99021, AR-A014418, TWS119, LY2090314, ING-41, The medium may include a basal medium (e.g., CTS OpTmizer, TexMACS, RPMI, DMEM, X-Vivo15, and others) containing supplements such as small molecules and additives, such as lithium chloride (LiCl), BIO (6-bromoindirubin-3-oxime, 6-bromoindirubin-3'-oxime), tyrosine kinase inhibitors (e.g., dasatinib, ibrutinib, acalabrutinib, and zanubrutinib), and caspase inhibitors (e.g., Emricasan, Z-VAD-FMK, Z-VKD-FMK, and others). At the end of stage 3 culture, the expanded TARGET cells can be harvested and formulated into a final TARGET cell product, which can be used fresh or cryopreserved for future use as an "off-the-shelf" supply.

[0041] In certain embodiments of the invention, an additional "CD4 induction step" can be added between stage 2 and stage 3 cultures to allow for the generation of CD4+ TARGET cells, and another additional "T H A "polarization step" is further added to stage 3 cultures to induce T H This allows for the generation of polarized CD4+ TARGET cells. In some embodiments, at the end of Stage 2 culture, once the developing TARGET cells reach the CD4+CD8+ double-positive (DP) stage, they can be induced to become CD4 SP TARGET cells by switching them to TARGETc-coated plates and culturing them in CD4 induction (CD4i) media for approximately 10-48 hours. CD4i Media can include TARGETm Media supplemented with T cell activation molecules (e.g., anti-CD3 / CD28 / CD2 antibodies, anti-CD3 / CD28 / CD2 beads, TCR stimulating antigens, phorbol 12-myristate 13-acetate [PMA] and ionomycin, phytohemagglutinin [PHA], and others). After this passage CD4 induction step, the developing TARGET cells can then be returned to fresh TARGETm Media and proceed with Stage 2 culture. In some embodiments, the induced CD4 SP TARGET cells are then induced to become "T" cells during Stage 3 culture. H T specified via the polarization step H In some embodiments, specific T H Polarizing reagents are added to the Stage 3 T cell culture medium to induce the designated T H It can produce CD4 SP target cells of the T subtype H 0 / T H 1-like TARGET cells (T H without adding any polarization reagent), T H TARGET cells like IL-12, IL-18, and anti-IL-4 antibodies H 1) by adding a polarization reagent, T H TARGET cells like IL-4 and anti-IFN-γ antibodies H 2 by adding a polarization reagent), T H17-like TARGET cells (TGF-β, IL-1β, IL-6, IL-21, IL-23, etc.) H 17) by adding a polarization reagent, T FH Target cells (TFs such as IL-12, including TGF-β or activin A) H by adding a polarization reagent), and T REG TARGET cells (e.g., IL-2 and TGF-β) REG At the end of Stage 3 culture, the resulting non-polarized or TH polarized CD4 SP TARGET cells can be harvested and formulated into a final TARGET cell product, which can be used fresh or cryopreserved for future use as an "off-the-shelf" supply.

[0042] In some embodiments, the TARGET cell product produced by the methods described herein can be cryopreserved. In some embodiments, the cryo-harvested cell product can be stable at room temperature for at least 1 hour. In some embodiments, the cryo-harvested cell product is stable at room temperature for at least 1, 2, 3, 4, 5, 6, 7, 8, 10, 15, 20, 24, 30, or 48 hours (or any derivable range therein). In certain embodiments, the cell product contains a solution comprising one or more of dextrose, electrolytes, albumin, dextran, and / or DMSO. In further embodiments, the cell product is in a solution that is sterile, non-purulent, and isotonic.

[0043]

[0010] Embodiments of the present invention include methods of generating monoclonal TCR-armed engineered T (TARGET) cells, comprising: (a) selecting T cell reprogrammed induced PSC (T-iPSC) cells comprising an endogenous T cell receptor (TCR); or (b) transducing pluripotent stem cells (e.g., from a PSC cell line) with at least one exogenous nucleic acid molecule encoding a TCR, such that cells transduced with the at least one exogenous nucleic acid molecule express a functional TCR encoded by the exogenous nucleic acid molecule; and differentiating the cells of (a) or (b) to generate monoclonal TCR-armed engineered T (TARGET) cells. Embodiments of the present invention further include monoclonal TCR-armed engineered T (TARGET) cells generated by the methods disclosed herein.

[0044] As described above, in certain embodiments of the present invention, pluripotent stem cells are genetically engineered via an all-in-one engineering (AO-Engineering) strategy, in which all genetic modifications are incorporated into a master PSC line. In alternative embodiments, pluripotent stem cells are genetically engineered via an assembly-line engineering (AL-Engineering) strategy, in which the desired genetic modifications are made stepwise on the master PSC line and its progeny hematopoietic stem and progenitor cells. In some embodiments of the present invention, the PSCs comprise T cell reprogrammed induced PSCs (T-iPSCs), and the TCR comprises an endogenous TCR. In some embodiments of the present invention, the genetic modifications are made before placing the PSC cells in differentiation medium. In certain embodiments of the present invention, the genetic modifications are made after placing the PSC cells in differentiation medium.

[0045]

[0010] An embodiment of the Assembly-Line of the present invention includes a method of generating monoclonal TCR-armed genetically engineered T (TARGET) cells, comprising: (a) placing pluripotent stem cells in a serum-free, feeder-free PSC culture medium comprising at least one of bFGF, TGFβ, FLT3L, Noggin, Activin, and Bio for at least 3, 6, or 12 hours; (b) combining the pluripotent stem cells from (a) with at least one exogenous nucleic acid molecule disposed in an expression vector and culturing the PSC cells for at least 3, 6, or 12 hours to identify pluripotent stem cells transduced with the expression vector; and (c) culturing the expression vector-transduced pluripotent stem cells from (b) that have been dissociated into single cells so that the cells form embryonic bodies. (d) placing the embryonic bodies of (c) in serum-free, feeder-free PSC differentiation culture medium B containing at least one of BMP-4, FGF, SCF, TPO, FLT3L, IL-6, IL-11, IGF-1, SB203580, and EPO for at least 2, 4, or 6 days to form CD34+ hematopoietic stem cells; and (e) placing the embryonic bodies of (c) in serum-free, feeder-free PSC differentiation culture medium B containing at least one of BMP-4, FGF, SCF, TPO, FLT3L, IL-6, IL-11, IGF-1, SB203580, and EPO for at least 3, 6, or 12 hours to form CD34+ hematopoietic stem cells. + (f) collecting and / or enriching hematopoietic stem cells from (e); and (f) CD34 + (g) placing the hematopoietic stem cells in a serum-free, feeder-free TARGET expansion cell culture medium containing at least one of serum albumin, recombinant human insulin, human transferrin, 2-mercaptoethanol, SCF, TPO, IL-3, IL-6, Flt3 ligand, human LDL, and UM171 for at least 1, 2, or 3 days; and (g) the CD34 +and placing the hematopoietic stem cells in a serum-free, feeder-free cell TARGET maturation medium containing at least one of serum albumin, recombinant human insulin, human transferrin, 2-mercaptoethanol, SCF, TPO, IL-3, IL-6, IL-7, IL-15, Flt3 ligand, and ascorbic acid, thereby generating monoclonal TCR-armed genetically engineered T (TARGET) cells. Some embodiments of the present invention further include placing the monoclonal TCR-armed genetically engineered T (TARGET) cells in a target cell growth medium containing at least one TCR cognate antigen or a nonspecific TCR stimulatory reagent. Optionally, the target cell growth medium includes feeder cells (e.g., a medium containing at least one of IL-2, IL-7, and IL-15). In some embodiments of this methodology, the method further includes cryopreserving the cells used in the methodology, e.g., the CD34+ hematopoietic stem cells generated in step (d).

[0046] In certain embodiments of the invention, the method generates TARGET cells that express at least 2,000 exogenous TCR polypeptides on the cell surface. In typical embodiments of the invention, the exogenous nucleic acid molecule encodes at least one T cell receptor selected from alpha beta TCR, gamma delta TCR, invariant NKT (TARGET) TCR, non-invariant NKT TCR, and mucosal-associated invariant TCR. In some embodiments of the invention, the exogenous nucleic acid molecule encoding the T cell receptor comprises a promoter selected for its ability to resist silencing in the TARGET cell, e.g., a human ubiquitin promoter. In some embodiments of the invention, the exogenous nucleic acid molecule is contained in a lentiviral expression vector and / or the exogenous nucleic acid molecule further encodes a polypeptide that stimulates T cells, a polypeptide that interferes with a T cell inhibitory factor, and / or a polypeptide comprising an additional receptor. Optionally, the polypeptide encoded by the exogenous nucleic acid comprises at least one of a chimeric antigen receptor (CAR), IL-2, IL-7, IL-15, IFN-γ, TNF-α, CD28, 4-1BB, OX40, ICOS, and FOXP3.

[0047] One exemplary embodiment of the present invention is a method for producing monoclonal TCR-armed engineered T (TARGET) cells, comprising transducing pluripotent stem cells with at least one exogenous nucleic acid molecule encoding a T cell receptor (TCR), wherein the exogenous nucleic acid molecule encodes at least one T cell receptor selected from the mucosal-associated invariant TCR alpha chain Va7.2-Ja33 / Ja20 / Ja12 and / or a restricted TCR beta chain, such that the transduced cells express a functional TCR encoded by the exogenous nucleic acid molecule; and differentiating the transduced cells to generate monoclonal TCR-armed engineered T (TARGET) cells. See, e.g., Li et al., Mol Ther. 2023 Mar 1;31(3):631-64. In certain of these embodiments, the transduced cells are cultured in a medium containing one or more of vitamin B2 or a vitamin B2 precursor (e.g., 5-(2-oxopropylideneamino)-6-D-ribitylaminouracil (5-OP-RU) and 5-(2-oxoethylideneamino)-6-D-ribitylaminouracil (5-OE-RU)); 5-amino-6-ribitylamino-2,4-(1H,3H)-pyrimidinedione (5-A-RU); a TLR9 agonist, IL-12 and / or IL-18, vitamin B2, or a vitamin B2 precursor.

[0048] Methods of treating a patient with a TARGET cell product are also provided. In certain embodiments, the patient has cancer. In other embodiments, the patient has a viral, bacterial, fungal, or parasitic infection. In some embodiments, the patient has a disease or condition involving inflammation, excluding cancer in some embodiments. In certain embodiments, the patient has an autoimmune disease or condition. In some embodiments, the TARGET cell product is allogeneic with respect to the patient. In further embodiments, the patient does not show signs of rejection or depletion of the TARGET cells. Some treatment methods further comprise administering to the patient a stimulatory reagent that activates the TARGET cells or a reagent that triggers a kill switch of a suicide gene.

[0049] Figures 1-26 collectively present data demonstrating the feasibility and therapeutic potential of the disclosed PSC-engineered TARGET cell technology. Various PSC resources (ESCs and various iPSCs, including T-iPSCs), genetic manipulation strategies (All-in-One and Assembly-Line Engineering strategies), genetic modifications (TCR, CAR, IL-15, reporter), and TARGET cell types (MAIT, iNKT, γδT, Tc) and subtypes (CD8 SP, CD4 SP, and Tc) are presented. H The CMC procedure is robust and the yield is impressive: 1 x 10 6 The input PSC is potentially about 2 × 10 6 CD34 HSPCs and ultimately approximately 10 13 These PSC-derived TARGET cells or their derivatives can be generated. The resulting PSC-derived TARGET cell products exhibit typical memory T cell and NK cell characteristics, potent in vitro and in vivo antitumor efficacy, and unique attributes such as low GvHD risk, resistance to allogeneic rejection, and the ability to alter the immunosuppressive TME. These unique attributes may be attractive for potential "off-the-shelf" cell therapy applications for a wide variety of diseases, including various cancers, particularly solid tumors, as well as infectious and autoimmune diseases.

[0050] FIG. 1 shows an overview of the PSC-T invention.

[0051] Figure 2 shows the successful generation of MAIT cell-reprogrammed iPSC (MAIT-iPSC) lines with high efficiency, as well as the validation and characterization of the resulting MAIT-iPSC lines.

[0052] Figure 3 shows the successful differentiation of various PSC sources into CD34+ HSPCs in scalable ex vivo feeder-free / serum-free cultures. PSCs tested include ESCs, fibroblast-reprogrammed iPSCs, CD34+ HSPC-reprogrammed iPSCs, conventional T cell-reprogrammed iPSCs, and MAIT cell-reprogrammed iPSCs. The resulting CD34+ HSPCs were differentiated at a high yield (1 x 10 6 Approximately 2 x 10 6 CD34+ HSPCs) and high purity (>60% CD34 + CD31 + CD45 + CD44 + CD43+CD144 - CD235a - ), eliminating the need for further purification. These CD34 HSPCs are also suitable for cryopreservation and may be useful for CMC development.

[0053] Figure 4 shows the successful differentiation of PSC-derived CD34+ HSPCs into T cells in a scalable ex vivo feeder-free / serum-free culture. Notably, when non-T-iPSC lines are used as starting PSCs, this PSC-HSPC differentiation culture can support successful rearrangement of endogenous TCR genes and the generation of both alpha-beta and gamma-delta T cells, highlighting the power and potential of this culture method.

[0054] Figure 5 shows the characterization of various PSC-derived T cells (differentiated from ESCs, T-iPSCs, and MAIT-iPSCs) and demonstrates their functionality as evidenced by significant proliferation in response to TCR antigen stimulation.

[0055] Figures 6 and 7 demonstrate the successful generation and characterization of PSC-derived MAIT cells and their CAR / IL-15-armed derivatives produced using an "All-in-One" Engineering (AO-Engineering) strategy. The various product designs tested all functioned robustly and with high yields.PSC15 The BCAR-MAIT cell product demonstrated robust in vitro tumor cell killing efficacy and utilization of a TCR / CAR / NKR triple targeting mechanism.

[0056] Figures 8-13 demonstrate the successful generation and characterization of PSC-derived MAIT cells and their CAR / IL-15-armed derivatives produced using an "Assembly-Line" Engineering (AL-Engineering) strategy. The various product designs tested all functioned robustly and with high yields. PSC15 The BCAR-MAIT cell product demonstrated robust antitumor efficacy and a TCR / CAR / NKR triple targeting mechanism. Importantly, AL manipulation PSC15 The BCAR-MAIT cell product demonstrated in vivo antitumor efficacy comparable to or better than that of conventional BCAR-T cells, as well as attractive attributes including GvHD-free risk, resistance to allogeneic rejection, and unique MAIT TCR-mediated TME targeting ability superior to conventional BCAR-T cells.

[0057] Figures 14 and 15 show PSC-derived CD4 single-positive (CD4 SP) MAIT cells and their T H2 The successful generation and characterization of polarized subtypes is demonstrated.

[0058] Figures 16-22 demonstrate the successful generation of PSC-derived iNKT cells and their CAR-armed derivatives and their potential as promising "off-the-shelf" cancer treatments.

[0059] Figures 23-26 demonstrate the successful generation of PSC-derived γδ T cells and their potential as a promising "off-the-shelf" cancer therapy.

[0060] The term "exogenous TCR" refers to a TCR gene or TCR gene derivative that is transferred (i.e., by gene transfer / transduction / transfection techniques) into a cell, or the progeny of a cell that has received the TCR gene or gene derivative. The exogenous TCR gene is inserted into the genome of the recipient cell. In some embodiments, the insertion is random insertion. Random insertion of a TCR gene is readily achieved by methods known in the art. In some embodiments, the TCR gene is inserted into an endogenous locus (such as an endogenous TCR locus). In some embodiments, the cell comprises one or more TCR genes inserted into a locus that is not an endogenous locus. In some embodiments, the cell further comprises a heterologous sequence, such as a marker or resistance gene.

[0061] The term "chimeric antigen receptor" or "CAR" refers to an engineered receptor that transfers any specificity onto immune effector cells. These receptors are used to transfer the specificity of a monoclonal antibody into T cells. The transfer of their coding sequences is facilitated by retroviral or lentiviral vectors. The receptors are called chimeric because they are composed of portions from different sources. The most common form of these molecules is a fusion of a single-chain variable fragment (scFv) derived from a monoclonal antibody fused to the CD3-zeta transmembrane domain and endodomain; the CD28 or 41BB intracellular domain; or a combination thereof. Such molecules result in signal transduction in response to recognition of their target by the scFv. An example of such a construct is 14g2a-ζ, a fusion of an scFv derived from the hybridoma 14g2a (which recognizes the disialoganglioside GD2). When T cells express this molecule (as achieved, for example, by oncoretroviral vector transduction), they recognize and kill target cells expressing GD2 (e.g., neuroblastoma cells). To target malignant B cells, researchers have redirected T cell specificity using chimeric immunoreceptors specific for the B lineage molecule, CD19. The variable portions of immunoglobulin heavy and light chains are fused by a flexible linker to form scFvs. This scFv is preceded by a signal peptide that directs the nascent protein to the endoplasmic reticulum and subsequent surface expression (which is cleaved). The flexible spacer allows the scFv to orient in different directions to enable antigen binding. The transmembrane domain is typically a typical hydrophobic alpha helix derived from the original molecule, which protrudes into the cell and transmits the desired signal.

[0062] The term "antigen" refers to any substance that can cause the immune system to produce antibodies against or T cells to respond to. In some embodiments, an antigen is a peptide that is 5-50 amino acids in length, or at least, at most, or exactly 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 125, 150, 175, 200, 250, or 300 amino acids, or any derivable range therein.

[0063] The term "allogeneic to the recipient" is intended to refer to cells that are not isolated from the recipient. In some embodiments, the cells are not isolated from the patient. In some embodiments, the cells are not isolated from a genetically matched individual (such as a relative with a compatible genotype).

[0064] The term "inactive" refers to not producing undesired clinical toxicity. This can be either on-target or off-target toxicity. "Inactive" can be based on known or predicted clinical safety data.

[0065] The terms "xeno-free (XF)" or "animal component-free (ACF)" or "animal-free," when used with reference to a medium, extracellular matrix, or culture condition, refer to a medium, extracellular matrix, or culture condition that is essentially free of xenogeneic animal-derived components. For culturing human cells, any protein from a non-human animal, such as a mouse, is a xenogeneic component. In certain embodiments, a xeno-free matrix is ​​essentially free of non-human animal-derived components and thus may exclude mouse feeder cells or Matrigel™. Matrigel™ is a solubilized basement membrane preparation extracted from the Engelbreth-Holm-Swarm (EHS) mouse sarcoma, a tumor enriched in extracellular matrix proteins, including laminin (the major component), type IV collagen, heparin sulfate proteoglycan, and entactin / nidogen.

[0066] The term "defined," when used in reference to a medium, extracellular matrix, or culture condition, refers to a medium, extracellular matrix, or culture condition in which the nature and amount of substantially all components are known.

[0067] "Chemically defined media" refers to media in which the chemical nature and amounts of nearly all components are known. These media are also called synthetic media. Examples of chemically defined media include TeSR™.

[0068] As used herein, cells are "substantially free" of a particular reagent or element, such as serum, signaling inhibitors, animal components or feeder cells, exogenous genetic elements or vector elements, if they contain less than 10% of the element, and "essentially free" of a particular reagent or element if they contain less than 1% of the element. However, even more desirable are cell populations in which less than 0.5% or less than 0.1% of the total cell population contains exogenous genetic elements or vector elements.

[0069] A culture, matrix, or medium is "essentially free" of a particular reagent or element, such as serum, signaling inhibitors, animal components, or feeder cells, if the culture, matrix, or medium has levels of these reagents below the level detectable using conventional detection methods known to those of skill in the art, respectively, or if these agents are not exogenously added to the culture, matrix, or medium. Serum-free medium may be essentially free of serum.

[0070] An embodiment of the present invention uses pluripotent stem "cell lines." As known in the art, a cell line is a cell culture derived from one cell or a set of cells of the same type, in which the cells grow indefinitely in the laboratory under specific conditions. In this way, cell lines differ from primary cells isolated from an individual in that they are immortalized. Cell lines further differ from primary cells isolated from an individual in that they are clonal (e.g., monoclonal or polyclonal). Because the physiology of cell lines differs from that of primary cells, those skilled in the art cannot predict how a cell line will behave solely from studying primary cells. In some embodiments of the invention, the pluripotent stem cell line is H1 (see, e.g., Getachew et al., Stem Cell Res. 2021 Jul;54:102401. In other embodiments of the invention, the pluripotent stem cell line is UCLA DMD1001R; UCLA iPS-21 stem cell line. Other exemplary stem cell lines are described, for example, in Sullivan et al., Regen Med. 2018 Oct;13(7):859-866. doi:10.2217 / rme-2018-0095; Capowski et al., Development. 2019 Jan 9;146(1); Ortman et al., Curr Opin Genet Dev. 2017 Oct;46:179-185; Kattman et al., Cell Stem Cell. 2011 Feb 4;8(2):228-40; Yu et al., Genes Dev. 2008 Aug 1;22(15):1987-97; Chhabra Stem Cell Rev Rep.2017 Dec;13(6):757-773; Stacey et al., Nucleic Acids Res.2016 Jan 4;44; and U.S. Patent Application Publication Nos. 20210310020, 20190153386, 20170226482, 20140154800, 20120083032, 20080311625, 20080267874, 20060160215, 20050095703, and 20030003088.

[0071] A "vector" or "construct" (sometimes called a gene delivery or gene transfer "vehicle") refers to a macromolecule, molecular complex, or viral particle containing a polynucleotide that is delivered to a host cell either in vitro or in vivo. The polynucleotide can be a linear or circular molecule.

[0072] A "plasmid," a common type of vector, is an extrachromosomal DNA molecule that can replicate independently of chromosomal DNA and is separate from chromosomal DNA. In certain cases, it is circular and double-stranded.

[0073] "Expression construct" or "expression cassette" means a nucleic acid molecule capable of directing transcription. An expression construct contains at least a promoter or a structure functionally equivalent to a promoter. Additional elements, such as an enhancer and / or a transcription termination signal, may also be included.

[0074] The term "exogenous," when used in reference to a protein, gene, nucleic acid, or polynucleotide in a cell or organism, refers to a protein, gene, nucleic acid, or polynucleotide that has been introduced into the cell or organism by artificial means, or, in reference to a cell, refers to a cell that has been isolated and subsequently introduced into another cell or organism by artificial means. An exogenous nucleic acid can be derived from a different organism or cell, or can be one or more additional copies of a nucleic acid that naturally occurs in the organism or cell. An exogenous cell can be derived from a different organism or from the same organism. As a non-limiting example, an exogenous nucleic acid is at a chromosomal location that is different from that of the native cell, or is flanked by different nucleic acid sequences than those found in nature.

[0075] The term "corresponding" is used herein to mean that a polynucleotide sequence is homologous (i.e., identical, not strictly evolutionarily related) to all or a portion of a reference polynucleotide sequence, or that a polypeptide sequence is identical to a reference polypeptide sequence. In contrast, the term "complementary" is used herein to mean that a complementary sequence is homologous to all or a portion of a reference polynucleotide sequence. For purposes of illustration, the nucleotide sequence "TATAC" corresponds to the reference sequence "TATAC" and is complementary to the reference sequence "GTATA."

[0076] A "gene," "polynucleotide," "coding region," "sequence," "segment," "fragment," or "transgene" "encoding" a particular protein is a nucleic acid molecule that, when placed under the control of appropriate regulatory sequences, can be transcribed in vitro or in vivo and, if necessary, translated into a gene product, e.g., a polypeptide. The coding region may be present in either cDNA, genomic DNA, or RNA form. If present in DNA form, the nucleic acid molecule may be single-stranded (i.e., the sense strand) or double-stranded. The boundaries of a coding region are determined by a start codon at the 5' (amino) terminus and a translation stop codon at the 3' (carboxy) terminus. A gene can include, but is not limited to, cDNA derived from prokaryotic or eukaryotic mRNA, genomic DNA derived from prokaryotic or eukaryotic DNA, and synthetic DNA sequences. A transcription termination sequence is typically located 3' to the gene sequence.

[0077] The term "cell" is used herein in the broadest sense in the art and refers to a structural unit of tissue in a multicellular organism, surrounded by a membrane structure that isolates it from the outside, capable of self-replication, and possessing genetic information and a mechanism for expressing it. As used herein, a cell may be a naturally occurring cell or an artificially modified cell (e.g., a fused cell, a genetically modified cell, etc.).

[0078] As used herein, the term "stem cell" refers to a cell that has self-renewal and pluripotency or multipotency. Typically, stem cells can regenerate damaged tissue. Stem cells in this specification can be, but are not limited to, embryonic stem (ES) cells, induced pluripotent stem cells or tissue stem cells (also called tissue-specific stem cells or somatic stem cells).

[0079] Embryonic stem (ES) cells are pluripotent stem cells derived from early embryos. ES cells were first established in 1981, and have been used to create knockout mice since 1989. Human ES cells were established in 1998, and are now available for use in regenerative medicine.

[0080] "Induced pluripotent stem cells," commonly abbreviated as iPS cells or iPSCs, refer to a type of pluripotent stem cell that is artificially prepared from non-pluripotent cells, typically adult somatic cells, or terminally differentiated cells such as fibroblasts, hematopoietic cells, muscle cells, neurons, and epidermal cells, by introducing specific factors called reprogramming factors.

[0081] As used herein, "isolated," for example with respect to cells and / or nucleic acids, means altered or removed from the natural state by human intervention.

[0082] "Pluripotent" refers to stem cells that have the potential to differentiate into all cells that make up one or more tissues or organs, particularly any of the three germ layers: endoderm (stomach lining, digestive tract, lungs), mesoderm (muscle, bone, blood, urogenital tract), or ectoderm (epidermal tissue and nervous system). As used herein, "pluripotent stem cells" refer to cells that can differentiate into cells derived from any of the three germ layers, e.g., the direct progeny of totipotent cells or induced pluripotent cells.

[0083] "Operably linked," with respect to nucleic acid molecules, means that two or more nucleic acid molecules (e.g., a transcribed nucleic acid molecule, a promoter, and an enhancer element) are linked in a manner that allows for transcription of the nucleic acid molecule. "Operably linked," with respect to peptide and / or polypeptide molecules, means that two or more peptide and / or polypeptide molecules are linked in a manner that results in a single polypeptide chain having at least one property of each peptide and / or polypeptide component of the fusion, i.e., a fusion polypeptide. Fusion polypeptides are particularly chimeric, i.e., composed of heterologous molecules.

[0084] Embodiments of the present disclosure relate to cells derived from pluripotent stem cells that have been engineered to function as target cells with T cell receptor (TCR), imaging and suicide targeting capabilities, and are resistant to host immune cell target depletion. Such cells are generated in a scalable PSC-T ex vivo culture system that supports the production of target cell products from pluripotent stem cells with high efficiency, high yield, and high purity.

[0085] In some embodiments, the engineered cells are functional TARGET cells, hi some embodiments, the engineered cells are capable of producing one or more cytokines and / or chemokines, such as IFN-gamma, TNF-alpha, TGF-beta, GM-CSF, IL-2, IL-4, IL-5, IL-6, IL-10, IL-13, IL-17, IL-21, RANTES, eotaxin, MIP-1-alpha, MIP-1-beta, etc.

[0086] In some embodiments, the TARGET cells from which the TCR-alpha chain is obtained and the TARGET cells from which the TCR-beta chain is obtained are derived from the same donor. In some embodiments, the donor of the TARGET cells from which the TCR-alpha chain is obtained is different from the donor of the TARGET cells from which the TCR-beta chain is obtained. In some embodiments, the TCR-alpha chain-encoding sequence and / or the TCR-beta chain-encoding sequence of a TCR clone are modified. In some embodiments, the modified sequence may encode the same polypeptide sequence as an unmodified TCR clone, e.g., the sequence is codon-optimized for expression. In some embodiments, the modified sequence may encode a polypeptide having a different sequence from the unmodified TCR clone, e.g., the modified sequence encodes one or more polypeptide sequences with amino acid substitutions, deletions, and / or truncations.

[0087] In certain embodiments, TARGET cells generated from pluripotent stem cells are further modified to have one or more characteristics, including making the cells suitable for allogeneic use or more suitable for allogeneic use than if the cells were not further modified to have one or more characteristics. The present disclosure optionally encompasses TARGET cells suitable for allogeneic use. In some embodiments, the TARGET cells are non-alloreactive and express an exogenous iNTK TCR. These cells are useful for "off-the-shelf" cell therapy, eliminating the need for the patient's own TARGET or other cells. Thus, the present method provides a more cost-effective and less labor-intensive cellular immunotherapy.

[0088] In specific embodiments, TARGET cells are engineered to be HLA-negative to achieve safe and successful allogeneic transplantation without causing graft-versus-host disease (GvHD) and without being rejected by host immune cells (HvG rejection). In certain embodiments, expression of the transgenic TARGET TCR gene blocks recombination of endogenous TCRs via allelic exclusion, so that allogeneic TARGET cells do not express endogenous TCRs and do not cause GvHD. In certain embodiments, allogeneic TARGET cells do not express HLA-I and / or HLA-II molecules on their cell surface and do not express host CD8 + and CD4 + Resist T cell-mediated allograft depletion and sr39TK immunogen target depletion.

[0089] Thus, in certain embodiments, the engineered TARGET cells do not express surface HLA-I or -II molecules, which is achieved by disruption of genes encoding proteins associated with HLA-I / II expression, including, but not limited to, beta2-microglobulin (B2M), major histocompatibility complex II transactivator (CIITA), or HLA-I / II molecules. In some cases, HLA-I or HLA-II is not expressed on the surface of the TARGET cells because the cells have been engineered by gene editing, which may or may not involve CRISPR-Cas9.

[0090] When a TARGET cell is modified to exhibit one or more characteristics of any kind, the TARGET cell may contain a nucleic acid sequence from a recombinant vector introduced into the cell. The vector may be a non-viral vector such as a plasmid, or a viral vector such as a lentivirus, retrovirus, adeno-associated virus (AAV), herpes virus, or adenovirus.

[0091] The TARGET cells of the present disclosure may or may not have been exposed to one or more specific conditions before, during, or after their production. In specific cases, the cells are not or have not been exposed to a medium containing animal serum. The cells may be frozen. The cells may be present in a solution containing dextrose, one or more electrolytes, albumin, dextran, and / or DMSO. Any solution in which the cells are present may be sterile, non-pyrogenic, and isotonic. The cells may be activated and expanded by any suitable method, such as by activation with alpha-galactosylceramide (α-GC).

[0092] Aspects of the present disclosure relate to human cells comprising: i) an exogenous expression or activity inhibitor of one or more of beta2 microglobulin (B2M), CIITA, TRAC, TRBC1, or TRBC2; or ii) a genomic mutation of one or more of beta2 microglobulin (B2M), CIITA, TRAC, TRBC1, or TRBC2. In some embodiments, the cell comprises a genomic mutation. In some embodiments, the genomic mutation comprises a mutation of one or more endogenous genes in the genome of the cell, wherein the one or more endogenous genes comprise B2M, CIITA, TRAC, TRBC1, or TRBC2 genes. In some embodiments, the mutation comprises a loss-of-function mutation. In some embodiments, the inhibitor is an expression inhibitor. In some embodiments, the inhibitor comprises an inhibitory nucleic acid. In some embodiments, the inhibitory nucleic acid comprises one or more of an siRNA, shRNA, miRNA, or antisense molecule. In some embodiments, the cell comprises an activity inhibitor. In some embodiments, after modification, the cells lack any detectable expression of one or more of the B2M, CIITA, TRAC, TRBC1, or TRBC2 proteins. In some embodiments, the cells comprise an inhibitor or genomic mutation of B2M. In some embodiments, the cells comprise an inhibitor or genomic mutation of CIITA. In some embodiments, the cells comprise an inhibitor or genomic mutation of TRAC. In some embodiments, the cells comprise an inhibitor or genomic mutation of TRBC1. In some embodiments, the cells comprise an inhibitor or genomic mutation of TRBC2. In some embodiments, at least 90% of the genomic DNA encoding B2M, CIITA, TRAC, TRBC1, and / or TRBC2 is deleted. In some embodiments, at least or at most 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 95, 99, or 100% (or any derivable range therein) of the genomic DNA encoding B2M, CIITA, TRAC, TRBC1, and / or TRBC2 is deleted. In other embodiments, deletions, insertions and / or substitutions are made in the genomic DNA. In some embodiments, the cells are the progeny of human stem or progenitor cells.

[0093] TARGET cells modified to be HLA-negative can be genetically modified by any suitable method. Gene mutations of the present disclosure, such as those in the CIITA and / or B2M genes, can be introduced by methods known in the art. In certain embodiments, engineered nucleases can be used to introduce exogenous nucleic acid sequences for genetic modification of any cell described herein. Genome editing, or genome editing with engineered nucleases (GEEN), is a type of genetic engineering in which DNA is inserted, replaced, or removed from a genome using artificially engineered nucleases, or "molecular scissors." The nucleases create specific double-strand breaks (DSBs) at desired locations within the genome, and the cell's endogenous machinery is used to repair the induced breaks through the natural processes of homologous recombination (HR) and non-homologous end joining (NHEJ). Non-limiting examples of engineered nucleases include zinc finger nucleases (ZFNs), TAL effector nucleases (TALENs), CRISPR / Cas9 systems, and engineered meganucleases and engineered homing endonucleases. Any engineered nuclease known in the art can be used in certain embodiments of the methods and compositions.

[0094] Engineered target cells can be modified using methods that employ RNA interference. To understand gene or protein function, sequence-specific interference and monitoring of the organism's effects are commonly practiced in genetic analysis. However, in some organisms, site-directed mutagenesis is difficult or impossible, and more indirect methods, such as silencing the gene of interest with short RNA interference (siRNA), must be used. However, siRNA-mediated gene disruption can be variable and incomplete. Genome editing with nucleases such as ZFNs differs from siRNA in that engineered nucleases can alter DNA binding specificity, thus cleaving any target location within the genome and introducing modifications to the endogenous sequence of genes that cannot be specifically targeted by conventional RNAi. Furthermore, the specificity of ZFNs and TALENs is enhanced because two ZFNs are required to recognize their portion of the target and then directly target adjacent sequences.

[0095] Meganucleases can be used to modify engineered target cells. Meganucleases commonly found in microbial species have the unique property of having very long recognition sequences (>14 bp), making them highly specific in nature. This can be exploited to create site-specific DSBs in genome editing. However, the challenge is that there may not be enough known or previously unknown meganucleases to cover all possible target sequences. To overcome this challenge, mutagenesis and high-throughput screening methods have been used to create meganuclease variants that recognize unique sequences. Others have fused various meganucleases to create hybrid enzymes that recognize new sequences. Still others have attempted to alter the DNA-interacting amino acids of meganucleases to engineer sequence-specific meganucleases, a method known as rationally designed meganucleases (U.S. Patent No. 8,021,867, incorporated herein by reference). Meganucleases have the advantage of causing less toxicity in cells compared to methods such as ZFNs, which are likely due to their more stringent DNA sequence recognition. However, constructing sequence-specific enzymes for every possible sequence is costly and time-consuming, and does not benefit from the combinatorial possibilities that methods such as ZFN and TALEN exploit. Thus, it has both advantages and disadvantages.

[0096] In contrast to meganucleases, the concept behind ZFNs and TALENs is based on nonspecific DNA-cleaving enzymes that are then linked to peptides that recognize specific DNA sequences, such as zinc fingers and transcription activator-like effectors (TALEs). One approach is to find an endonuclease whose DNA recognition and cleavage sites are separated from each other, a situation that is uncommon among restriction enzymes. Once this enzyme is found, its cleavage portion can be separated, but it is highly nonspecific because it has no recognition ability. This portion can then be linked to a sequence-recognition peptide, which can result in extremely high specificity. An example of a restriction enzyme with such properties is FokI. Furthermore, FokI has the advantage of requiring dimerization to have nuclease activity, meaning that specificity increases dramatically as each nuclease partner recognizes a unique DNA sequence. To enhance this effect, FokI nucleases have been engineered to function only as heterodimers and have increased catalytic activity. Heterodimeric nucleases avoid the possibility of undesired homodimer activity and therefore increase the specificity of DSBs.

[0097] While the nuclease moieties of both ZFNs and TALENs share similar properties, the difference between these engineered nucleases lies in their DNA recognition peptides. ZFNs rely on Cys2-His2 zinc fingers, while TALENs rely on TALEs. Both of these DNA recognition peptide domains are unique in that they are naturally found in combination within proteins. Cys2-His2 zinc fingers typically occur in repeats spaced 3 bp apart and are found in diverse combinations within various nucleic acid-interacting proteins, such as transcription factors. TALEs, on the other hand, are found in repeats with a one-to-one recognition ratio between the amino acid and the recognized nucleotide pair. Because both zinc fingers and TALEs occur in a repetitive pattern, different combinations can be attempted to create a wide variety of sequence specificities. Zinc fingers are more established in these terms, and approaches such as modular assembly (zinc fingers correlating with triplet sequences are linked in a line to cover the required sequence), OPEN (low stringency selection of peptide domains versus triplet nucleotides, followed by high stringency selection of peptide combinations against the final target in bacterial systems), and bacterial one-hybrid screening of zinc finger libraries, among other methods, have been used to create site-specific nucleases.

[0098] Thus, embodiments of the present disclosure may or may not involve targeting of endogenous sequences to reduce or knock out expression of one or more specific endogenous sequences. In certain embodiments, disruption of one or more of the following genes may block endogenous TCR rearrangement:

[0099] Inhibitory nucleic acids or any method known in the art for inhibiting gene expression of CIITA and / or B2M are contemplated in certain embodiments. Examples of inhibitory nucleic acids include, but are not limited to, siRNA (small interfering RNA), short hairpin RNA (shRNA), double-stranded RNA, antisense oligonucleotides, ribozymes, and nucleic acids encoding them. Inhibitory nucleic acids can inhibit gene transcription or prevent translation of gene transcripts in cells. Inhibitory nucleic acids can be 16 to 1000 nucleotides in length, and in certain embodiments, 18 to 100 nucleotides in length. Nucleic acid can have at least or at most 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 40, 50, 60, 70, 80, 90 or any range of nucleotides that can be derived therefrom.Silicone that naturally exists in living animals is not "isolated", but synthetic siRNA or siRNA that is partially or completely separated from the coexisting materials in its natural state is "isolated".Isolated siRNA can exist in substantially purified form or can exist in a non-natural environment (for example, the cell into which siRNA is delivered).

[0100] Inhibitory nucleic acid is well known in the art.For example, siRNA and double-stranded RNA are described in United States Patent (USP) No. 6,506,559 and No. 6,573,099, and United States Patent Application Publication No. 2003 / 0051263, No. 2003 / 0055020, No. 2004 / 0265839, No. 2002 / 0168707, No. 2003 / 0159161 and No. 2004 / 0064842, all of which are incorporated herein by reference in their entirety.

[0101] In particular, the inhibitory nucleic acid can reduce protein or mRNA expression by at least 10%, 20%, 30% or 40%, more particularly by at least 50%, 60% or 70%, and most particularly by at least 75%, 80%, 90%, 95% or more, or any range or value therebetween.

[0102] In further embodiments, there are synthetic nucleic acids that are protein inhibitors. The inhibitors can be 17-25 nucleotides in length and contain a 5' to 3' sequence that is at least 90% complementary to the 5' to 3' sequence of a mature mRNA. In certain embodiments, the inhibitor molecule is 17, 18, 19, 20, 21, 22, 23, 24, or 25 nucleotides in length, or any derivable range therein. Furthermore, the inhibitor molecule has a sequence (5' to 3') that is at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 99.1, 99.2, 99.3, 99.4, 99.5, 99.6, 99.7, 99.8, 99.9, or 100% complementary to the 5' to 3' sequence of a mature, naturally occurring mRNA, such as the mRNA for B2M, CIITA, TRAC, TRBC1, or TRBC2, or any derivable range therein. Those skilled in the art can use the portion of the probe sequence that is complementary to the sequence of the mature mRNA as the sequence of the mRNA inhibitor, and furthermore, that portion of the probe sequence can be altered so that it is still 90% complementary to the sequence of the mature mRNA.

[0103] When the engineered TARGET cells contain one or more suicide genes for subsequent depletion as needed, the suicide genes may be of any suitable type. The TARGET cells of the present disclosure may express a suicide gene product, which may be, for example, enzyme-based. Examples of suicide gene products include herpes simplex virus thymidine kinase (HSV-TK), purine nucleoside phosphorylase (PNP), cytosine deaminase (CD), carboxypeptidase G2, cytochrome P450, linamarase, beta-lactamase, nitroreductase (NTR), carboxypeptidase A, or inducible caspase 9. Thus, in a specific case, the suicide gene may encode thymidine kinase (TK). In a specific case, the TK gene is a viral TK gene, such as the herpes simplex virus TK gene. In certain embodiments, the suicide gene product is activated by a substrate such as ganciclovir, penciclovir, or a derivative thereof.

[0104] In some embodiments, the engineered TARGET cells can be imaged or otherwise detected. In certain cases, the cells contain an exogenous nucleic acid encoding a polypeptide with a substrate that can be labeled for imaging, and imaging can be fluorescent, radioactive, colorimetric, or the like. In specific cases, the cells are detected by positron emission tomography. In at least some cases, the cells express the sr39TK gene, a positron emission tomography (PET) reporter / thymidine kinase gene, allowing these genetically modified cells to be tracked by PET imaging and eliminated by the sr39TK suicide gene function.

[0105] A population of engineered TARGET cells is encompassed by the present disclosure. In certain embodiments, the TARGET clonal cells contain an exogenous nucleic acid encoding a TARGET T cell receptor (T cell receptor) and lack surface expression of one or more HLA-I or HLA-II molecules. The TARGET cells may contain an exogenous nucleic acid encoding a suicide gene, including an enzyme-based suicide gene such as thymidine kinase (TK). The TK gene may be a viral TK gene, such as the herpes simplex virus TK gene. In the cells of the population, the suicide gene can be activated by a substrate, such as ganciclovir, penciclovir, or a derivative thereof. The cells may contain an exogenous nucleic acid encoding a polypeptide having a substrate that can be labeled for imaging; in some cases, the suicide gene product is a polypeptide having a substrate that can be labeled for imaging. In a specific embodiment, the suicide gene is sr39TK.

[0106] In certain embodiments of the TARGET cell population, the expression of genes encoding, for example, beta 2 microglobulin (B2M), major histocompatibility complex class II transcription activator (CIITA), and / or HLA-I or HLA-II molecules is disrupted, so that the TARGET cells do not express surface HLA-I or HLA-II molecules. HLA-I or HLA-II molecules are not expressed on the cell surface of the TARGET cells because the cells are engineered by gene editing in certain cases. Gene editing may or may not involve CRISPR-Cas9.

[0107] In certain cases of the TARGET cell population, the TARGET cells comprise nucleic acid sequences from a recombinant vector that has been introduced into the cells, such as a viral vector (including at least a lentivirus, retrovirus, adeno-associated virus (AAV), herpesvirus, or adenovirus).

[0108] In certain embodiments, the cells of the TARGET cell population may or may not have been exposed to one or more specific conditions, or may be exposed to one or more specific conditions. In certain cases, for example, the cells of the population are not or have not been exposed to a medium containing animal serum. The cells of the population may or may not be frozen. In some cases, the cells of the population are present in a solution containing dextrose, one or more electrolytes, albumin, dextran, and / or DMSO. The solution may contain dextrose, one or more electrolytes, albumin, dextran, and DMSO. The cells may be present in a solution that is sterile, non-pyrogenic, and isotonic. In specific cases, the TARGET cells are activated, such as activated with alpha-galactosylceramide (α-GC). In certain aspects, the cell population is at least about 10 2 ~10 6 The cell population optionally comprises at least about 10 clonal cells. 6 ~10 13 The total number of cells may be 100.

[0109] In certain embodiments, there is a TARGET cell population comprising clonal TARGET cells comprising a TARGET T cell receptor (T cell receptor) and one or more exogenous nucleic acids encoding thymidine kinase suicide, wherein the clonal TARGET cells have been engineered not to express functional beta 2 microglobulin (B2M), major histocompatibility complex class II transactivator (CIITA), and / or HLA-I and HLA-II molecules, and the cell population comprises at least about 10 total cells. 6 ~10 13 At least about 10 2 ~10 6 Optionally, the cells are frozen in solution.

[0110] II. Cell Formulation and Culture In certain embodiments, TARGET cells and / or their precursors may be specifically constructed and / or cultured in a particular medium at any stage of the process to generate the TARGET cells. The cells may be constructed to be suitable for delivery to a recipient without adverse effects.

[0111] In certain embodiments, the medium can be prepared using any of media used to culture animal cells, such as AIMV, X-VIVO-15, NeuroBasal, EGM2, TeSR, BME, BGJb, CMRL1066, Glasgow MEM, Improved MEM Zinc Option, IMDM, Medium 199, Eagle MEM, αMEM, DMEM, Ham, RPMI-1640, and Fischer's medium, as well as any combination thereof, as its basal medium, but is not limited thereto as long as it can be used to culture animal cells. In particular, the medium may be xeno-free or chemically defined.

[0112] The medium may be serum-containing or serum-free, or xeno-free. To prevent contamination with components derived from different animals, the serum may be derived from the same animal as the stem cells. Serum-free medium refers to a medium that does not contain untreated or unpurified serum, and therefore may include a medium containing purified blood-derived components or animal tissue-derived components (such as growth factors).

[0113] The medium may or may not contain a serum replacement. Serum replacements include materials that suitably contain albumin (e.g., lipid-rich albumin, bovine albumin, albumin substitutes such as recombinant or humanized albumin, plant starch, dextran, and protein hydrolysates), transferrin (or other iron transporters), fatty acids, insulin, collagen precursors, trace elements, 2-mercaptoethanol, 3'-thioglycerol, or equivalents thereof. Serum replacements can be prepared, for example, by the methods disclosed in International Publication No. 98 / 30679 (incorporated herein in its entirety). Alternatively, for greater convenience, any commercially available material can be used. Commercially available materials include knockout serum replacement (KSR), chemically-defined lipid concentrated (Gibco), and Glutamax (Gibco).

[0114] In further embodiments, the medium may be a serum-free medium suitable for cell development. For example, the medium may contain B-27® supplement, Xenofree B-27® supplement (available on the World Wide Web at thermofisher.com / us / en / home / technical-resources / media-formulation.250.html), NS21 supplement (Chen et al., J Neurosci Methods, 2008 Jun 30;171(2):239-247, incorporated herein in its entirety), GS21™ supplement (available on the World Wide Web at amsbio.com / B-27.aspx), or a combination thereof, at a concentration effective to generate T cells from the 3D cell aggregates.

[0115] In certain embodiments, the medium may contain one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, nineteen, twenty or more of the following: vitamins such as biotin; DL alpha tocopherol acetate; DL alpha-tocopherol; vitamin A (acetate); proteins such as BSA (bovine serum albumin) or human albumin, fatty acid free fraction V; catalase; human recombinant insulin; human transferrin; superoxide dismutase; other components such as corticosterone; D-galactose; ethanolamine HCl; glutathione (reduced); L-carnitine HCl; linoleic acid; linolenic acid; progesterone; putrescine 2HCl; sodium selenite; and / or T3 (triiodo-I-thyronine).

[0116] In some embodiments, the medium further comprises vitamins. In some embodiments, the medium comprises one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, or thirteen (and any range derivable therein) of biotin, DL-alpha tocopherol acetate, DL-alpha-tocopherol, vitamin A, choline chloride, calcium pantothenate, pantothenic acid, folic acid nicotinamide, pyridoxine, riboflavin, thiamine, inositol, vitamin B12, or a combination thereof or a salt thereof, or the medium comprises a combination thereof or a salt thereof. In some embodiments, the medium comprises or consists essentially of biotin, DL-alpha tocopherol acetate, DL-alpha-tocopherol, vitamin A, choline chloride, calcium pantothenate, pantothenic acid, folic acid nicotinamide, pyridoxine, riboflavin, thiamine, inositol, and vitamin B12. In some embodiments, the vitamin comprises or consists essentially of biotin, DL-alpha tocopherol acetate, DL-alpha-tocopherol, vitamin A, or a combination or salt thereof. In some embodiments, the medium further comprises a protein. In some embodiments, the protein comprises albumin or bovine serum albumin, a fraction of BSA, catalase, insulin, transferrin, superoxide dismutase, or a combination thereof. In some embodiments, the medium further comprises one or more of corticosterone, D-galactose, ethanolamine, glutathione, L-carnitine, linoleic acid, linolenic acid, progesterone, putrescine, sodium selenite, or triiodo-I-thyronine, or a combination thereof. In some embodiments, the medium comprises one or more of B-27® supplement, XenoFree B-27® supplement, GS21™ supplement, or a combination thereof. In some embodiments, the medium comprises or further comprises amino acids, simple sugars, and inorganic ions.In some embodiments, the amino acids include arginine, cystine, isoleucine, leucine, lysine, methionine, glutamine, phenylalanine, threonine, tryptophan, histidine, tyrosine, or valine, or a combination thereof. In some embodiments, the inorganic ions include sodium, potassium, calcium, magnesium, nitrogen, or phosphorus, or a combination or salt thereof. In some embodiments, the medium further includes one or more of molybdenum, vanadium, iron, zinc, selenium, copper, or manganese, or a combination thereof. In certain embodiments, the medium comprises or consists essentially of one or more of the vitamins discussed herein and / or one or more of the proteins discussed herein, and / or one or more of the following: corticosterone, D-galactose, ethanolamine, glutathione, L-carnitine, linoleic acid, linolenic acid, progesterone, putrescine, sodium selenite or triiodo-I-thyronine, B-27® supplement, XenoFree B-27® supplement, GS21™ supplement, amino acids (e.g., arginine, cystine, isoleucine, leucine, lysine, methionine, glutamine, phenylalanine, threonine, tryptophan, histidine, tyrosine, or valine), monosaccharides, inorganic ions (e.g., sodium, potassium, calcium, magnesium, nitrogen, and / or phosphorus, etc.) or salts thereof, and / or molybdenum, vanadium, iron, zinc, selenium, copper, or manganese.

[0117] In further embodiments, the medium may include exogenously added ascorbic acid. The medium may also contain one or more of exogenously added fatty acids or lipids, amino acids (such as non-essential amino acids), vitamins, growth factors, cytokines, antioxidants, 2-mercaptoethanol, pyruvic acid, buffers, and / or inorganic salts.

[0118] One or more media components may be added at a concentration of at least, at most, or about 0.1, 0.5, 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150, 180, 200, 250 ng / L, ng / ml, μg / ml, mg / ml, or any range derivable therein.

[0119] The medium used may be supplemented with at least one exogenously added cytokine at a concentration of about 0.1 ng / mL to about 500 ng / mL, more particularly 1 ng / mL to 100 ng / mL, or at least, at most, or about 0.1, 0.5, 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150, 180, 200, 250 ng / L, ng / ml, μg / ml, mg / ml, or any range inducible therein. Suitable cytokines include, but are not limited to, FLT3 ligand (FLT3L), interleukin 7 (IL-7), stem cell factor (SCF), thrombopoietin (TPO), IL-2, IL-4, IL-6, IL-15, IL-21, TNF-alpha, TGF-beta, interferon-gamma, interferon-lambda, TSLP, thymopentin, pleiotrophin, and / or myosin. In particular, the medium may contain at least one of FLT3L and IL-7. More specifically, the culture may contain both FLT3L and IL-7.

[0120] Other culture conditions can be determined as appropriate. For example, the culture temperature can be about 20 to 40°C, e.g., at least, at most, or about 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40°C (or any inducible range therein), although the temperature can be higher or lower than these values. The CO2 concentration can be about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10% (or any inducible range therein), e.g., about 2% to 10%, e.g., about 2 to 5%, or any inducible range therein. The oxygen tension can be at least or about 1, 5, 8, 10, or 20%, or any inducible range therein.

[0121] In specific embodiments, the allogeneic HSC-manipulated HLA-negative TARGET cells are specifically formulated. They may or may not be formulated as a cell suspension. In certain cases, they are formulated in a single-dose form. They may be formulated for systemic or local administration. In some cases, the cells are formulated for storage before use, and the cell formulation may include one or more cryopreservatives, such as DMSO (e.g., in 5% DMSO). The cell formulation may include albumin, including human albumin. A specific formulation includes 2.5% human albumin. The cells may be specifically formulated for intravenous administration. For example, they are formulated for intravenous administration over a period of less than one hour. In certain embodiments, the cells are in a formulated cell suspension that is stable at room temperature for 1, 2, 3, or 4 hours or more from the time of thawing.

[0122] In some embodiments, the method further comprises priming the TARGET cells. In some embodiments, the TARGET cells are primed with antigen-presenting cells. In some embodiments, the antigen-presenting cells present a tumor antigen.

[0123] In certain embodiments, the exogenous TCR of the TARGET cell can be of any defined antigen specificity. In some embodiments, it can be selected based on the lack or reduced alloreactivity to the intended recipient (examples include a specific virus-specific TCR, a xenospecific TCR, or a cancer-testis antigen-specific TCR). In instances where the exogenous TCR is non-alloreactive, during T cell differentiation, the exogenous TCR suppresses rearrangement and / or expression of the endogenous TCR locus through a developmental process called allelic exclusion, resulting in T cells that express only the non-alloreactive exogenous TCR and are therefore non-alloreactive. In some embodiments, the selection of the exogenous TCR does not necessarily have to be defined based on the lack of alloreactivity. In some embodiments, the endogenous TCR gene is modified by genome editing so that it does not express the protein. Methods of gene editing, such as those using the CRISPR / Cas9 system, are known in the art and are described herein.

[0124] In some embodiments, the isolated TARGET cells or populations thereof comprise one or more chimeric antigen receptors (CARs). Examples of tumor cell antigens to which CARs can be directed include at least 5T4, 8H9, α vβ6 integrin, BCMA, B7-H3, B7-H6, CAIX, CA9, CD19, CD20, CD22, CD30, CD33, CD38, CD44, CD44v6, CD44v7 / 8, CD70, CD123, CD138, CD171, CEA, CSPG4, EGFR, EGFR family including ErbB2 (HER2), EGFRvIII, EGP2, EGP40, ERBB3, ERBB4, ErbB3 / 4, EPCAM, EphA2, EpCAM, folate receptor-α, FAP, FBP, fetal AchR, FRα, GD2, G250 / CAIX, GD3, glypican-3 (GPC3), Her2, IL-13Rα2, lambda, Lewis Y, kappa, KDR, MAGE, MCSP, mesothelin, Muc1, Muc16, NCAM, NKG2D ligand, NY-ESO-1, PRAME, PSC1, PSCA, PSMA, ROR1, SP17, survivin, TAG72, TEM, carcinoembryonic antigen, HMW-MAA, AFP, CA-125, ETA, tyrosinase, MAGE, laminin receptor, HPV CARs include E6, E7, BING-4, calcium-activated chloride channel 2, cyclin-B1, 9D7, EphA3, telomerase, SAP-1, BAGE family, CAGE family, GAGE ​​family, MAGE family, SAGE family, XAGE family, NY-ESO-1 / LAGE-1, PAME, SSX-2, Melan-A / MART-1, GP100 / pmel17, TRP-1 / -2, P. polypeptide, MC1R, prostate-specific antigen, β-catenin, BRCA1 / 2, CML66, fibronectin, MART-2, TGF-βRII, or VEGF receptor (e.g., VEGFR2). CARs can be first-, second-, third-, or higher-generation CARs. CARs can be bispecific for any two non-identical antigens, or specific for three or more non-identical antigens.

[0125] III. Further Modifications and Polypeptide Embodiments Additionally, the polypeptides of the present disclosure may be chemically modified. The glycosylation of the polypeptide can be altered, for example, by modifying one or more sites of glycosylation within the polypeptide sequence to increase the affinity of the polypeptide for an antigen (U.S. Patent Nos. 5,714,350, 6,350,861).

[0126] Substitutional variants typically involve exchanging one amino acid for another at one or more sites within the protein and can be designed to modulate one or more properties of the polypeptide, with or without loss of other functions or properties. Substitutions can be conservative, i.e., one amino acid is replaced with one of similar shape and charge. Conservative substitutions are well known in the art, and include, for example, alanine to serine; arginine to lysine; asparagine to glutamine or histidine; aspartic acid to glutamic acid; cysteine ​​to serine; glutamine to asparagine; glutamic acid to aspartic acid; glycine to proline; histidine to asparagine or glutamine; isoleucine to leucine or valine; leucine to valine or isoleucine; lysine to arginine; methionine to leucine or isoleucine; phenylalanine to tyrosine, leucine or methionine; serine to threonine; threonine to serine; tryptophan to tyrosine; tyrosine to tryptophan or phenylalanine; valine to isoleucine or leucine. Alternatively, substitutions can be non-conservative, such that the function or activity of the polypeptide is affected. Non-conservative changes typically involve substituting a residue with a chemically different residue, e.g., a polar or charged amino acid for a non-polar or uncharged amino acid, or vice versa.

[0127] The protein may be recombinant or synthesized in vitro. Alternatively, non-recombinant or recombinant proteins may be isolated from bacteria. It is also contemplated that bacteria containing such mutants may be used in compositions and methods. Thus, the protein does not need to be isolated.

[0128] The term "functionally equivalent codon" is used herein to refer to codons that encode the same amino acid, such as the six codons for arginine or serine, and also refers to codons that encode biologically equivalent amino acids.

[0129] It will also be understood that amino acid and nucleic acid sequences may include additional residues, such as additional N- or C-terminal amino acids, or 5' or 3' sequences, respectively, and still be essentially as set forth in one of the sequences disclosed herein, so long as the sequence meets the above criteria, including maintenance of the biological protein activity to which protein expression pertains. The addition of terminal sequences particularly applies to nucleic acid sequences, which may include, for example, various non-coding sequences adjacent to either the 5' or 3' portion of the coding region.

[0130] The following is a consideration based on the amino acid modification of proteins to create equivalent or further improved second-generation molecules.For example, certain amino acids can be substituted with other amino acids in protein structures without obvious loss of interactive binding ability.For example, structures such as enzyme catalytic domains or interaction components can have substituted amino acids to maintain such functions.Because the interaction ability and properties of proteins define the biological functional activity of proteins, certain amino acid substitutions can be made in protein sequences and their underlying DNA coding sequences, and still produce proteins with similar properties.Therefore, the inventors believe that various changes can be made to the DNA sequence of genes without obvious loss of biological usefulness or activity.

[0131] In other embodiments, polypeptide alterations are intended by introducing one or more substitutions. For example, specific amino acids can be substituted with other amino acids in the protein structure to alter the interactive binding ability of interacting components. For example, structures such as protein interaction domains, nucleic acid interaction domains, and catalytic sites can have substituted amino acids to change their functions. Because the interaction ability and properties of a protein determine the biological functional activity of a protein, specific amino acid substitutions can be made in the protein sequence and its underlying DNA coding sequence, and still produce proteins with different properties. Therefore, the present inventors contemplate that various changes can be made to the DNA sequence of a gene, with significant changes in biological usefulness or activity.

[0132] In making such changes, the hydropathic index of amino acids may be taken into consideration. The importance of the hydropathic amino acid index in conferring interactive biological function on a protein is generally understood in the art (Kyte and Doolittle, 1982). It is recognized that the relative hydropathic properties of amino acids contribute to the secondary structure of the resulting protein, which in turn defines the interactions of the protein with other molecules, such as enzymes, substrates, receptors, DNA, antibodies, antigens, etc.

[0133] It is also understood in the art that similar amino acids can be effectively substituted based on hydrophilicity. U.S. Patent No. 4,554,101, incorporated herein by reference, states that the greatest local average hydrophilicity of a protein, as governed by the hydrophilicity of adjacent amino acids, correlates with the biological properties of the protein. It is understood that an amino acid can be substituted with another having a similar hydrophilicity value to produce a biologically equivalent and immunologically equivalent protein.

[0134] As outlined above, amino acid substitutions are generally based on the relative similarity of the amino acid side-chain substituents, e.g., their hydrophobicity, hydrophilicity, charge, size, etc. Exemplary substitutions that take into account various of the foregoing characteristics are well known and include arginine and lysine; glutamic acid and aspartic acid; serine and threonine; glutamine and asparagine; valine, leucine, and isoleucine.

[0135] In certain embodiments, all or part of the proteins described herein can be synthesized in solution or on a solid support according to conventional techniques. A variety of automated synthesizers are commercially available and can be used according to known protocols. See, for example, Stewart and Young, (1984); Tam et al., (1983); Merrifield, (1986); and Barany and Merrifield (1979), each of which is incorporated herein by reference. Alternatively, peptides or polypeptides can be synthesized using recombinant DNA technology, in which the encoding nucleotide sequence is inserted into an expression vector, transformed or transfected into a suitable host cell, and cultured under conditions suitable for expression.

[0136] One embodiment involves the use of gene transfer into cells, including microorganisms, for protein production and / or display. The gene for the protein of interest is transferred into a suitable host cell, which can then be cultured under appropriate conditions. Nucleic acids encoding virtually any polypeptide can be used. The generation of recombinant expression vectors and the elements contained therein are discussed herein. Alternatively, the protein produced can be an endogenous protein normally synthesized by the cells used for protein production.

[0137] IV. Method for producing TARGET cells The examples provided herein are merely exemplary TARGET cells, and TARGET cells can be produced by a variety of suitable methods. The methods may utilize one or more sequential steps for one or more modifications to the cells, and / or one or more simultaneous steps for one or more modifications to the cells. In certain embodiments, a starting source of cells from a cell line is modified to function as TARGET cells, followed by one or more steps that impart one or more additional characteristics to the cells, such as the ability to be imaged, and / or the ability to be selectively killed, and / or the ability to be used in an allogeneic system. In certain embodiments, at least a portion of the process for generating TARGET cells is carried out in a specific in vitro culture system. Examples of specific in vitro culture systems are those that allow for high-efficiency and high-yield differentiation of specific cells.

[0138] In specific cases, TARGET cells can be generated by: 1) genetically modifying cells from a pluripotent stem cell line to express a TARGET TCR (e.g., via a lentiviral vector) and, optionally, to eliminate expression of HLA-I / II molecules (e.g., via CRISPR / Cas9-based gene editing); 2) in vitro differentiation into TARGET cells via culture; 3) in vitro purification and expansion of TARGET cells; and 4) construction and cryopreservation and / or use.

[0139] Certain aspects of the present disclosure relate to novel three-dimensional cell culture systems for producing TARGET cells from less differentiated cell lines, such as embryonic stem cell lines, pluripotent stem cell lines, hematopoietic stem or progenitor cell lines, induced pluripotent stem (iPS) cell lines, or stem or progenitor cell lines.

[0140] In certain embodiments, the system includes using a serum-free medium. In certain aspects, the system uses a serum-free medium suitable for cell development for culturing the three-dimensional cell aggregates. Such a system generates sufficient amounts of TARGET cells. In embodiments of the present disclosure, the 3D cell aggregates are cultured in a serum-free medium containing insulin for a period sufficient for in vitro differentiation of stem cells or progenitor cells into TARGET cells, or precursors into TARGET cells.

[0141]

[0003] Embodiments of cell culture compositions include those that use highly standardized serum-free components and stromal cell lines to promote robust and highly reproducible T cell differentiation from human HSCs. In certain embodiments, cell differentiation mimics endogenous thymocyte formation and, in contrast to monolayer co-cultures, supports efficient positive selection of functional TARGET cells. Certain aspects of the 3D culture compositions use serum-free conditions, avoiding the use of human thymus tissue or proprietary scaffold materials, and promote positive selection and robust generation of fully functional, mature human TARGET cells from source cells.

[0142] In a method for preparing a population of clonal TARGET cells, selecting TARGET cells lacking surface expression of HLA-I and HLA-II molecules can include contacting the TARGET cells with magnetic beads that bind to the TARGET cells, positively selecting them, and negatively selecting HLA-I / II-negative cells. In certain embodiments, the magnetic beads are coated with monoclonal antibodies that recognize human TARGET TCR, HLA-I molecules, or HLA-II molecules. In certain embodiments, the monoclonal antibodies are Clone 6B11 (which recognizes human TCR Vα24-Jα18 and thus recognizes the human TARGET invariant TCR alpha chain), Clone 2M2 (which recognizes human B2M and thus recognizes cell surface-displayed human HLA-I molecules), Clone W6 / 32 (which recognizes HLA-A, B, C and therefore recognizes human HLA-I molecules), and Clone Tu39 (which recognizes human HLA-DR, DP, DQ and therefore recognizes human HLA-II molecules).

[0143] The cells produced by the preparation method can be frozen. The cells can be in a solution containing dextrose, one or more electrolytes, albumin, dextran, and DMSO. The solution can be sterile, non-pyrogenic, and isotonic.

[0144] In certain embodiments, the system comprises: - Feeder cells are utilized that may contain the cells.

[0145] The preparation method can further include activating and expanding the selected TARGET cells. For example, the selected TARGET cells are activated with alpha-galactosylceramide (α-GC). The feeder cells can be pulsed with α-GC.

[0146] The preparation method of the present disclosure comprises at least about 10 2 ~10 6 This method can produce a population of clonal TARGET cells comprising at least about 10 clonal TARGET cells. 6 ~10 12 A cell population containing a total of 100 cells can be produced. The produced cell population can be frozen and then thawed. In some cases, the preparation method further comprises introducing one or more additional nucleic acids, such as one or more additional nucleic acids encoding one or more therapeutic gene products, into the frozen and thawed cell population.

[0147] For example, aggregation can be achieved by centrifugation of the mixed cell suspension ("compression aggregation") followed by aspiration of the cell-free supernatant. In certain embodiments, the cell pellet can then be aspirated as a slurry in 5-10 μl of differentiation medium and transferred as a droplet onto a 0.4 μm nylon transwell culture insert, which floats within the well of differentiation medium, with the bottom of the insert in contact with the medium and the top in contact with air.

[0148] Variations in the protocol allow for the use of alternative ingredients that have different effects on efficacy, specifically:

[0149] The basal medium RPMI can be used in place of some commercially available alternatives (eg, IMDM).

[0150] The stromal cell line used is MS-5, a previously described murine bone marrow cell line (Itoh et al., 1989), although MS-5 can be substituted for similar murine stromal cell lines (e.g., OP9, S17), human stromal cell lines (e.g., HS-5, HS-27a), primary human stromal cells, or human pluripotent stem cell-derived stromal cells.

[0151] Stromal cell lines can be transduced with lentivirus encoding human DLL1 cDNA. However, the method of gene delivery, as well as the Notch ligand gene, can vary. Alternative Notch ligand genes include DLL4, JAG1, JAG2, and others. Notch ligands also include those described in U.S. Patent Nos. 7,795,404 and 8,377,886, which are incorporated herein by reference. Notch ligands further include Delta 1, 3, and 4, and Jagged 1 and 2.

[0152] Cytokine conditions can be varied: for example, FLT3L and IL-7 levels can be altered to alter T cell differentiation kinetics. Other hematopoietic cytokines, such as stem cell factor (SCF / KIT ligand), thrombopoietin (TPO), IL-2, and IL-15, may also be added.

[0153] Genetic modifications can also be introduced into specific components to generate antigen-specific T cells and model positive and negative selection. Examples of these modifications include transducing HSCs with lentiviral vectors encoding antigen-specific T cell receptors (TCRs) or chimeric antigen receptors (CARs) to generate antigen-specific, allelically excluded naive T cells, and transducing HSCs with genes to direct lineage commitment to specific lymphoid cells. For example, transducing HSCs with invariant natural killer T cell (TARGET)-associated TCRs to generate functional TARGET cells, transducing cells with human MHC genes (e.g., human CD1d genes) to enhance the positive selection and maturation of both TCR-engineered and non-TCR-engineered T cells, and / or transducing cell lines with antigens plus costimulatory molecules or cytokines to enhance the positive selection of CAR T cells.

[0154] When generating engineered target cells, CD34 + Cells can be modified by introducing specific exogenous genes and knocking out specific endogenous genes. This method involves introducing CD34 selected in culture medium prior to introducing one or more nucleic acids into the cells. + The cell line may further comprise culturing the cells. The culturing may comprise culturing CD34 cells selected in a medium containing one or more growth factors. + This can include incubating the cells with, optionally, one or more growth factors, which can include, for example, c-kit ligand, flt-3 ligand, and / or human thrombopoietin (TPO), at a particular concentration, e.g., from about 5 ng / ml to about 500 ng / ml.

[0155] In certain methods, the nucleic acid introduced into the cell is one or more nucleic acids containing nucleic acid sequences encoding α-TCR and β-TCR. The method may include introducing a nucleic acid encoding a suicide gene into the selected cell. In certain embodiments, one nucleic acid encodes both α-TCR and β-TCR, or one nucleic acid encodes α-TCR, β-TCR, and the suicide gene. The suicide gene may be enzyme-based, such as thymidine kinase (TK), including viral TK genes, such as those derived from herpes simplex virus TK genes. The suicide gene may be activated by substrates such as ganciclovir, penciclovir, or derivatives thereof. The cell may be engineered to contain an exogenous nucleic acid encoding a polypeptide having a substrate that can be labeled for imaging. In some cases, the suicide gene product is a polypeptide having a substrate that can be labeled for imaging, such as sr39TK.

[0156] Cells can be engineered to lack surface expression of HLA-I and / or HLA-II molecules, for example, by disrupting the functional expression of genes encoding beta2 microglobulin (B2M), major histocompatibility complex class II transcription activator (CIITA), and / or HLA-I and HLA-II molecules. In the above-described production method, eliminating surface expression of one or more HLA-I / II molecules in isolated human CD34+ cells can include introducing CRISPR and one or more guide RNAs (gRNAs) corresponding to B2M, CIITA, or individual HLA-I or HLA-II molecules into the cells. The CRISPR or one or more gRNAs are transfected into the cells, optionally by electroporation or lipid-mediated transfection. In certain embodiments, the nucleic acid encoding the TCR receptor is introduced into the cells using a recombinant vector, such as a viral vector comprising at least a lentivirus, retrovirus, adeno-associated virus (AAV), herpesvirus, or adenovirus.

[0157] In producing engineered TARGET cells, the cells may be present in certain serum-free media, including those containing exogenously added ascorbic acid. In certain embodiments, the serum-free media further contains exogenously added FLT3 ligand (FLT3L), interleukin 7 (IL-7), stem cell factor (SCF), thrombopoietin (TPO), IL-2, IL-4, IL-6, IL-15, IL-21, TNF-alpha, TGF-beta, interferon-gamma, interferon-lambda, TSLP, thymopentin, pleotrophin, or midkine, or a combination thereof. The serum-free medium may further contain vitamins, including biotin, DL-alpha tocopherol acetate, DL-alpha-tocopherol, vitamin A, choline chloride, calcium pantothenate, pantothenic acid, folic acid nicotinamide, pyridoxine, riboflavin, thiamine, inositol, vitamin B12, or combinations thereof or salts thereof. The serum-free medium may further contain one or more exogenously added (or non-added) proteins, such as albumin or bovine serum albumin, a fraction of BSA, catalase, insulin, transferrin, superoxide dismutase, or combinations thereof. The serum-free medium may further contain corticosterone, D-galactose, ethanolamine, glutathione, L-carnitine, linoleic acid, linolenic acid, progesterone, putrescine, sodium selenite, or triiodo-I-thyronine, or combinations thereof. The serum-free medium may contain B-27® supplement, Xenofree B-27® supplement, GS21™ supplement, or a combination thereof. Amino acids (including arginine, cysteine, isoleucine, leucine, lysine, methionine, glutamine, phenylalanine, threonine, tryptophan, histidine, tyrosine, or valine, or a combination thereof), simple sugars, and / or inorganic ions (including, for example, sodium, potassium, calcium, magnesium, nitrogen, or phosphorus, or a combination or salt thereof) may be present in the serum-free medium.The serum-free medium may further comprise molybdenum, vanadium, iron, zinc, selenium, copper, or manganese, or a combination thereof.

[0158] Cell culture conditions can be provided for culturing the 3D cell aggregates described herein and for producing and / or positively / negatively selecting T cells. In certain embodiments, the starting cells of the selected population are at least or about 10 4 , 10 5 , 10 6 , 10 7 , 10 8 , 10 9 , 10 10 , 10 11 , 10 12 , 10 13 The starting cell population may comprise at least or about 10, 10 1 , 10 2 , 10 3 , 10 4 , 10 5 , 10 6 , 10 7 , 10 8 The seeding density may be 100 cells / ml, or any range derivable therein.

[0159] Culture vessels used to culture 3D cell aggregates or their progeny cells can include, but are not limited to, flasks, tissue culture flasks, dishes, Petri dishes, tissue culture dishes, multi-dishes, microplates, microwell plates, multi-plates, multi-well plates, microslides, chamber slides, tubes, trays, CellSTACK® Chambers, culture bags, and roller bottles, as long as they are capable of culturing stem cells. Depending on the needs of the culture, stem cells can be cultured in volumes of at least or about 0.2, 0.5, 1, 2, 5, 10, 20, 30, 40, 50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 800, 1000, 1500 ml, or any range derivable therein. In certain embodiments, the culture vessel may be a bioreactor, which can refer to any device or system that supports a biologically active environment. The bioreactor can have a volume of at least or about 2, 4, 5, 6, 8, 10, 15, 20, 25, 50, 75, 100, 150, 200, 500 liters, 1, 2, 4, 6, 8, 10, 15 cubic meters, or any range derivable therein.

[0160] The culture vessel can be cell-adhesive or non-adhesive, and can be selected according to the purpose. The cell-adhesive culture vessel can be coated with a substrate for cell adhesion, such as an extracellular matrix (ECM), to improve the adhesion of the vessel surface to cells. The substrate for cell adhesion can be any material intended to adhere stem cells or feeder cells (if used). Substrates for cell adhesion include collagen, gelatin, poly-L-lysine, poly-D-lysine, laminin, and fibronectin, as well as mixtures thereof, such as Matrigel™, and dissolved cell membrane preparations.

[0161] A variety of defined matrix components can be used in the culture methods or compositions. For example, as described in Ludwig et al. (2006a; 2006b), which are incorporated by reference in their entireties, recombinant collagen IV, fibronectin, laminin, and vitronectin can be used in combination to coat a culture surface as a means of providing a solid support for pluripotent cell growth.

[0162] A matrix composition can be immobilized on a surface to provide support for cells. The matrix composition can include one or more extracellular matrix (ECM) proteins and an aqueous solvent. The term "extracellular matrix" is recognized in the art. Its components include one or more of the following proteins: fibronectin, laminin, vitronectin, tenascin, entactin, thrombospondin, elastin, gelatin, collagen, fibrillin, merosin, anchorin, chondronectin, link protein, bone sialoprotein, osteocalcin, osteopontin, epinectin, hyaluronectin, andurin, epiligrin, and kalinin. Other extracellular matrix proteins are described in Kleinman et al. (1993), which is incorporated herein by reference. The term "extracellular matrix" is intended to encompass currently unknown extracellular matrices that may be discovered in the future, as their characterization as extracellular matrices can be readily determined by those skilled in the art.

[0163] In some aspects, the total protein concentration in the matrix composition can be about 1 ng / mL to about 1 mg / mL. In some embodiments, the total protein concentration in the matrix composition is about 1 μg / mL to about 300 μg / mL. In more preferred embodiments, the total protein concentration in the matrix composition is about 5 μg / mL to about 200 μg / mL.

[0164] Extracellular matrix (ECM) proteins can be of natural origin and purified from human or animal tissues. Alternatively, ECM proteins can be genetically engineered recombinant proteins or essentially artificial. ECM proteins can be in the form of natural or engineered whole proteins or peptide fragments. Examples of ECM proteins that can be useful in matrices for cell culture include laminin, collagen I, collagen IV, fibronectin, and vitronectin. In some embodiments, the matrix composition comprises synthetically produced peptide fragments of fibronectin or recombinant fibronectin.

[0165] In still further embodiments, the matrix composition comprises a mixture of at least fibronectin and vitronectin. In some other embodiments, the matrix composition preferably comprises laminin.

[0166] The matrix composition preferably contains a single type of extracellular matrix protein. In some embodiments, the matrix composition contains fibronectin, specifically for use in culturing progenitor cells. For example, a suitable matrix composition can be prepared by diluting human fibronectin, such as that sold by Becton, Dickinson & Co. (BD) of Franklin Lakes, New Jersey (catalog number 354008), in Dulbecco's phosphate-buffered saline (DPBS) to a protein concentration of 5 μg / mL to approximately 200 μg / mL. In a specific example, the matrix composition contains a fibronectin fragment, such as RetroNectin®. RetroNectin® is a 574-amino acid, approximately 63 kDa protein containing a central cell-binding domain (type III repeats 8, 9, and 10), a high-affinity heparin-binding domain II (type III repeats 12, 13, and 14), and the CS1 site within the alternatively spliced ​​IIICs region of human fibronectin.

[0167] In some other embodiments, the matrix composition may include laminin. For example, a suitable matrix composition may be prepared by diluting laminin (Sigma-Aldrich, St. Louis, MO; catalog numbers L6274 and L2020) in Dulbecco's phosphate-buffered saline (DPBS) to a protein concentration of 5 μg / ml to about 200 μg / ml.

[0168] In some embodiments, the matrix composition is xeno-free, meaning that the matrix or its component proteins are exclusively of human origin. This may be desirable for certain research applications. For example, a xeno-free matrix for culturing human cells can use matrix components of human origin and exclude any non-human animal components. In certain aspects, Matrigel™ can be excluded as a substrate from the culture composition. Matrigel™ is a gelatinous protein mixture secreted by mouse tumor cells and is commercially available from BD Biosciences (New Jersey, USA). This mixture resembles the complex extracellular environment found in many tissues and is frequently used by cell biologists as a substrate for cell culture, but it can introduce unwanted xenoantigens or contaminants.

[0169] In certain embodiments, cells containing exogenous nucleic acid can be identified in vitro or in vivo by including a marker in the expression vector or exogenous nucleic acid. Such a marker will confer a identifiable change to the cell, allowing for easy identification of cells containing the expression vector. Generally, a selectable marker can confer a characteristic that allows selection. A positive selectable marker can be a marker whose presence allows its selection, while a negative selectable marker is a marker whose presence prevents its selection. An example of a positive selectable marker is a drug resistance marker.

[0170] Typically, the inclusion of a drug selection marker aids in the cloning and identification of transformants, e.g., genes conferring resistance to neomycin, puromycin, hygromycin, DHFR, GPT, zeocin, and histidinol. In addition to markers that confer a phenotype that allows for the identification of transformants based on the implementation of a condition, other types of markers are contemplated, including screenable markers such as GFP, whose basis is colorimetric analysis. Alternatively, screenable enzymes such as herpes simplex virus thymidine kinase (tk) or chloramphenicol acetyltransferase (CAT) may be utilized as negative selection markers. Those skilled in the art will likely know how to use immunological markers in conjunction with FACS analysis. The marker used is not considered critical, so long as it can be expressed simultaneously with the nucleic acid encoding the gene product. Further examples of selectable and screenable markers are well known to those skilled in the art.

[0171] Selectable markers can include the type of reporter gene used in laboratory microbiology, molecular biology, and genetic engineering to indicate the success of transfection or other procedures intended to introduce foreign DNA into cells. Selectable markers are often antibiotic resistance genes; cells engineered to introduce foreign DNA are grown on media containing the antibiotic; cells that are able to grow indicate that they have successfully taken up and expressed the introduced genetic material. Examples of selectable markers include the Abicr gene from Tn5, which confers antibiotic resistance to Geneticin, or the Neo gene.

[0172] Screenable markers may include reporter genes, which allow researchers to distinguish between desired and undesired cells. Certain embodiments of the present invention utilize reporter genes to indicate specific cell lineages. For example, reporter genes may be located within expression elements under the control of ventricular- or atrial-selective regulatory elements normally associated with the coding region of ventricular- or atrial-selective genes for coexpression. Reporters allow cells of specific lineages to be isolated without placing them under drug or other selective pressure or jeopardizing cell viability.

[0173] Examples of such reporters include genes encoding cell surface proteins (e.g., CD4, HA epitope), fluorescent proteins, antigenic determinants, and enzymes (e.g., β-galactosidase). Cells containing the vector can be isolated, for example, by FACS using fluorescently tagged antibodies against cell surface proteins or substrates that can be converted to fluorescent products by vector-encoded enzymes.

[0174] In certain embodiments, the reporter gene is a fluorescent protein. A wide range of fluorescent protein genetic variants have been developed, featuring fluorescent emission spectral profiles spanning nearly the entire visible light spectrum. Mutagenesis attempts on the original Aequorea victoria jellyfish green fluorescent protein have resulted in new fluorescent probes ranging from blue to yellow, and are some of the most widely used in vivo reporter molecules in biological research. Longer wavelength fluorescent proteins emitting in the orange and red spectral regions have been developed from the marine sea anemone Discosoma striata, belonging to the class Anthozoa, and reef coral. Still other species have been mined to produce similar proteins with cyan, green, yellow, orange, and crimson fluorescent emissions. Development research efforts are underway to improve the brightness and stability of fluorescent proteins, and thus their overall utility.

[0175] In certain embodiments, cells can be engineered to contain one or more genetic alterations by genetic manipulation of cells either before or after differentiation (US Patent Application Publication No. 2002 / 0168766). When an exogenous nucleic acid or polynucleotide is transferred into a cell by any suitable means of artificial manipulation, or when the cell is a descendant of an originally engineered cell that inherits the polynucleotide, the cell is said to be "genetically modified," "genetically modified," or "transgenic." For example, cells can be engineered to increase their replicative potential by genetically modifying the cells to express telomerase reverse transcriptase either before or after progressing to a restricted developmental lineage cell or a terminally differentiated cell (US Patent Application Publication No. 2003 / 0022367).

[0176] In certain embodiments, cells containing an exogenous nucleic acid construct can be identified in vitro or in vivo by including a marker, such as a selectable or screenable marker, in the expression vector. Such markers confer identifiable changes to cells that allow for easy identification of cells containing the expression vector, or aid in enriching or identifying differentiated cardiac cells by using tissue-specific promoters. For example, in cardiomyocyte differentiation, cardiac-specific promoters such as those for cardiac troponin I (cTnI), cardiac troponin T (cTnT), sarcomeric myosin heavy chain (MHC), GATA-4, Nkx2.5, N-cadherin, α1-adrenergic receptor, ANF, the MEF-2 family of transcription factors, creatine kinase MB (CK-MB), myoglobin, or atrial natriuretic factor (ANF) may be used. In neuronal differentiation, neuron-specific promoters may be used, including, but not limited to, TuJ-1, Map-2, Dcx, or synapsin. In hepatocyte differentiation embodiments, definitive endoderm and / or hepatocyte specific promoters may be used, including but not limited to ATT, Cyp3a4, ASGPR, FoxA2, ​​HNF4a, or AFP.

[0177] Generally, a selectable marker is one that confers a property that allows for selection. A positive selectable marker is one whose presence allows for its selection, while a negative selectable marker is one whose presence prevents its selection. An example of a positive selectable marker is a drug resistance marker.

[0178] Typically, the inclusion of a drug selectable marker aids in the cloning and identification of transformants; for example, genes conferring resistance to blasticidin, neomycin, puromycin, hygromycin, DHFR, GPT, zeocin, and histidinol are useful selectable markers. In addition to markers that confer a phenotype that allows for the identification of transformants based on the implementation of a condition, other types of markers are contemplated, including screenable markers based on colorimetric analysis, such as GFP. Alternatively, screenable enzymes such as chloramphenicol acetyltransferase (CAT) may be utilized. Those skilled in the art will likely know how to use immunological markers in conjunction with FACS analysis. The marker used is not considered critical, so long as it can be expressed simultaneously with the nucleic acid encoding the gene product. Further examples of selectable and screenable markers are well known to those skilled in the art.

[0179] In the embodiment in which cells are genetically modified to add or reduce one or more characteristics, genetic modification can be carried out by any suitable method.For example, any genetic modification composition or method can be used to introduce exogenous nucleic acid into cells or edit genomic DNA, such as gene editing, homologous recombination or non-homologous recombination, RNA-mediated gene delivery or any conventional nucleic acid delivery method.Non-limiting examples of genetic modification methods can include gene editing methods such as CRISPR / CAS9, zinc finger nuclease or TALEN technology.

[0180] Genetic modification can also include the introduction of selectable or screenable markers to aid in in vitro or in vivo selection, screening, or imaging. In particular, in vivo imaging agents or suicide genes can be exogenously expressed or added to the starting cells or progeny cells. In a further aspect, the method can include image-guided adoptive cell therapy.

[0181] V. How to Use the Cells The TARGET cells of the present disclosure may or may not be utilized immediately after production. In some cases, they are preserved for later use. In either case, they may be utilized for therapeutic or prophylactic purposes in mammalian subjects, such as patients (humans, dogs, cats, horses, etc.). Patients may require cell therapy for any type of medical condition, including allogeneic cell therapy.

[0182] The disclosed method of treating a patient with a therapeutically effective amount of TARGET cells includes administering the cells or a clonal population thereof to the patient. The cells or cell population can be allogeneic to the patient. In certain embodiments, the patient does not show signs of depletion of the cells or cell population. The patient may or may not have a disease or condition involving cancer and / or inflammation. In certain embodiments, the patient has cancer, tumor cells in the cancer patient die after administering the cells or cell population to the patient. In certain cases, the patient has inflammation, the inflammation is alleviated after administering the cells or cell population to the patient. In certain embodiments of the treatment method, the method further includes administering to the patient a compound that induces a suicide gene product.

[0183] In patients with cancer, it is anticipated that this cell product, when infused into the patient, will be able to use multiple mechanisms to target and eradicate tumor cells. +They can directly recognize and kill tumor cells. They secrete cytokines such as IFN-γ, which activate cells to kill HLA-negative tumor cells and DCs, which then stimulate cytotoxic T cells to kill HLA-positive tumor cells. Therefore, we plan a series of in vitro and in vivo studies to demonstrate the pharmacological efficacy of this cell product for cancer treatment.

[0184] Because TARGET cells can target a wide range of cancers without tumor antigen and MHC restrictions, ready-made TARGET cell products are useful as general cancer immunotherapy for treating any type of cancer and a large group of cancer patients.In particular, this treatment is useful for patients with cancers that have been clinically shown to be amenable to TARGET cell modulation, including, for example, multiple types of solid tumors (melanoma, colon cancer, lung cancer, breast cancer and head and neck cancer) and blood cancers (leukemia, multiple myeloma and myelodysplastic syndrome).

[0185] In some embodiments of any of the methods disclosed above, the subject has or is at risk of having an autoimmune disease, graft-versus-host disease (GVHD), or graft rejection. The subject may be a subject diagnosed with such a disease or a subject determined to have a predisposition to such a disease based on genetic or family history analysis. The subject may also be a subject preparing for or having undergone a transplant. In some embodiments, the method is for treating an autoimmune disease, GVHD, or graft rejection.

[0186] Individuals treated with the cell therapy may or may not have been treated for a particular medical condition prior to receiving the TARGET cell therapy. If the individual has cancer, the cancer may be primary, metastatic, refractory, etc. Patients who have exhausted conventional treatment options.

[0187] In certain embodiments, the cells are administered at a concentration of 10 per dose. 7 ~10 9In a specific embodiment, the administration regimen is a single dose of allogeneic TARGET cells after lymphocyte-depleting conditioning. The cells can be administered intravenously, for example, after lymphocyte-depleting conditioning with fludarabine and cyclophosphamide.

[0188] When in vivo antitumor efficacy was characterized for subsequent in vivo treatment cases, the in vivo pharmacological response was assessed by administering increasing doses (1x10) of EGFR to tumor-bearing NSG mice. 6 , 5x10 6 , 10x10 6 ) TARGET cells (n=8 per group). Treatment with PBS may be included as a control. As an example, two tumor models may be utilized: A375.CD1d (1x10 6 subcutaneous) may be used as a solid tumor model, and MM.1S.Luc (5x10 6 Intravenous (IV) transfection may be used as a hematological malignancy model. Tumor growth can be monitored by measuring either size (A375.CD1d) or bioluminescence imaging (MM.1S.Luc). Antitumor immune responses can be measured by PET imaging, periodic bleeding, and endpoint tumor harvest followed by flow cytometry and qPCR. Inhibition of tumor growth in response to TARGET treatment can indicate the therapeutic efficacy of TARGET cell therapy. Correlation between tumor inhibition and TARGET dose can confirm the therapeutic role of TARGET cells and indicate an effective therapeutic window for human treatment. Detection of TARGET cell responses against tumors can demonstrate the pharmacological antitumor activity of these cells in vivo.

[0189] The methods can be used with individuals who have tested positive for a medical condition, who have one or more symptoms of a medical condition, or who are considered to be at risk for developing such a condition. In some embodiments, the compositions and methods described herein are used to treat inflammatory or autoimmune components of disorders listed herein and / or known in the art.

[0190] Certain aspects of the present disclosure relate to the treatment of cancer and / or the use of cancer antigens. The cancer or antigen to be treated can be any cancer-associated antigen known in the art, or, for example, epithelial cancer (e.g., breast, gastrointestinal, lung), prostate cancer, bladder cancer, lung (e.g., small cell lung) cancer, colon cancer, ovarian cancer, brain cancer, stomach cancer, renal cell carcinoma, pancreatic cancer, liver cancer, esophageal cancer, head and neck cancer, or colorectal cancer. In some embodiments, the cancer or antigen being treated is from one of the following cancers: adrenocortical carcinoma, primary myelofibrosis, AIDS-related cancer (e.g., AIDS-related lymphoma), anal cancer, appendiceal cancer, astrocytoma (e.g., cerebellar and cerebral), basal cell carcinoma, bile duct cancer (e.g., extrahepatic), bladder cancer, bone cancer (osteosarcoma and malignant fibrous histiocytoma), brain tumor (e.g., glioma, brain stem glioma), cerebellar or cerebral astrocytoma (e.g., pilocytic astrocytoma, diffuse astrocytoma). , anaplastic (malignant) astrocytoma), malignant glioma, ependymoma, oligodendroglioma, meningioma, meningeal sarcoma, craniopharyngioma, hemangioblastoma, medulloblastoma, supratentorial primitive neuroectodermal tumor, visual pathway and hypothalamic glioma, glioblastoma), breast cancer, bronchial adenoma / carcinoid, carcinoid tumor (e.g., gastrointestinal carcinoid tumor), cancer of unknown primary, central nervous system lymphoma, cervical cancer, colon cancer, colorectal cancer, chronic myeloproliferative disorder, endometrial cancer (e.g., uterine cancer), ependymoma, esophageal Cancer, Ewing's family of tumors, eye cancer (e.g., intraocular melanoma and retinoblastoma), gallbladder cancer, gastric cancer, gastrointestinal carcinoid tumors, gastrointestinal stromal tumors (GIST), germ cell tumors (e.g., extracranial, extragonadal, ovarian), gestational trophoblastic neoplasia, head and neck cancer, hepatocellular (liver) cancer (e.g., hepatocarcinoma and hepatocellular carcinoma), hypopharyngeal cancer, islet cell carcinoma (endocrine pancreas), laryngeal cancer, leukemia, lip and oral cavity cancer, oral cancer, liver cancer, lung cancer (e.g., small cell lung cancer, non-small cell lung cancer), Lung cancer, lung adenocarcinoma, lung squamous cell carcinoma, etc.), lymphoid tumors (e.g., lymphoma), medulloblastoma, ovarian cancer, mesothelioma, metastatic squamous cell neck cancer, oral cancer, multiple endocrine neoplasia syndrome, myelodysplastic syndrome, myelodysplastic / myeloproliferative disorders, nasal cavity and paranasal sinus cancer, nasopharyngeal cancer, neuroblastoma, neuroendocrine cancer, oropharyngeal cancer, ovarian cancer (e.g., ovarian epithelial cancer, ovarian germ cell tumor, ovarian low malignant potential tumor), pancreatic cancer, parathyroid cancer, penile cancer, peritoneal cancer, pharyngeal cancer, pheochromocytoma,These include, but are not limited to, pineoblastoma and supratentorial primitive neuroectodermal tumor, pituitary tumor, pleuropulmonary blastoma, lymphoma, primary central nervous system lymphoma (microglioma), pulmonary lymphangioleiomyomatosis, rectal cancer, kidney cancer, renal pelvis and ureter cancer (transitional cell carcinoma), rhabdomyosarcoma, salivary gland cancer, skin cancer (e.g., non-melanoma (e.g., squamous cell carcinoma), melanoma, Merkel cell carcinoma), small intestine cancer, squamous cell carcinoma, testicular cancer, pharyngeal cancer, thymoma and thymic carcinoma, thyroid cancer, tuberous sclerosis complex, urethral cancer, vaginal cancer, vulvar cancer, Wilms' tumor, post-transplant lymphoproliferative disorder (PTLD), abnormal blood vessel proliferation associated with nevus syndrome, edema (such as that associated with a brain tumor), or Meigs' syndrome.

[0191] Certain aspects of the present disclosure relate to the treatment of autoimmune conditions and / or the use of autoimmune-associated antigens. The autoimmune disease or antigen to be treated can be an antigen associated with any autoimmune condition known in the art, or can be an antigen associated with, for example, diabetes, transplant rejection, GVHC, arthritis (rheumatoid arthritis, e.g., acute arthritis, chronic rheumatoid arthritis, gout or gouty arthritis, acute gouty arthritis, acute immunological arthritis, chronic inflammatory arthritis, degenerative arthritis, type II collagen-induced arthritis, infectious arthritis, Lyme arthritis, proliferative arthritis, psoriatic arthritis, Still's disease, spondyloarthritis, and juvenile-onset rheumatoid arthritis, osteoarthritis, chronic proarthritis, osteo ... psoriasis, such as psoriasis vulgaris, psoriasis of the gut, pustular psoriasis, and psoriasis of the nail; atopy, including atopic diseases such as hay fever and Jobs' syndrome; dermatitis, including contact dermatitis; chronic contact dermatitis; exfoliative dermatitis; allergic dermatitis; allergic contact dermatitis; dermatitis herpetiformis; dermatitis, including myriad dermatitis, seborrheic dermatitis, nonspecific dermatitis, primary irritant contact dermatitis, and atopic dermatitis; X-linked hyper IgM syndrome; allergic eye disease; Inflammatory diseases, chronic autoimmune urticaria such as chronic allergic urticaria and chronic idiopathic urticaria, myositis, scleroderma (including systemic sclerosis), sclerosis, e.g. systemic sclerosis, multiple sclerosis (MS), e.g. spinal MS, primary progressive MS (PPMS) and relapsing-remitting MS (RRMS), progressive systemic sclerosis, atherosclerosis, arteriosclerosis, disseminated sclerosis, ataxic sclerosis, neuromyelitis optica (NMO), inflammatory bowel disease (IBD) (e.g. Crohn's disease, autoimmune-mediated gastrointestinal diseases), ulcerative colitis, microscopic colitis, including idiopathic colitis, collagenous colitis, polyposous colitis, necrotizing enterocolitis and transmural colitis, and autoimmune inflammatory bowel diseases), intestinal inflammation, pyoderma gangrenosum, erythema nodosum, primary sclerosing cholangitis, respiratory distress syndromes including adult or acute respiratory distress syndrome (ARDS), meningitis, inflammation of all or part of the uvea, iritis, choroiditis, autoimmune blood disorders, rheumatoid spondylitis, rheumatoid synovitis, hereditary angioedema, e.g., herpes gestationis, pemphigus gestationis, vitiligo, premature meningitis, autoimmune ovarian failure, sudden hearing loss due to autoimmune conditions,IgE-mediated diseases such as anaphylaxis and allergic rhinitis and atopic rhinitis, encephalitis such as Rasmussen's encephalitis and limbic encephalitis and / or brainstem encephalitis, uveitis such as anterior uveitis, acute anterior uveitis, granulomatous uveitis, non-granulomatous uveitis, phacoantigenic uveitis, posterior uveitis or autoimmune uveitis, with or without renal syndromes such as chronic or acute glomerulonephritis, including primary GN Glomerulonephritis (GN), immune-mediated GN, membranous GN (membranous nephropathy), idiopathic membranous GN or idiopathic membranous nephropathy, membranous or membranous proliferative GN (MPGN) including types I and II, as well as rapidly progressive GN, proliferative nephritis, autoimmune polyendocrinopathy, perioningitis, leptomeningeal inflammation, erythema annulare centrifugally, erythema dyschromicum, erythema multiforme, granuloma annulare, melasma, lichen sclerosus / lichen atrophicus, lichen simplex, lichen spinous, lichen planus, lamellar ichthyosis, and epidermal keratosis. , premalignant keratosis, pyoderma gangrenosum, allergic conditions and reactions, eczema including allergic reactions, allergic or atopic eczema, asteatotic eczema, dyshidrotic eczema and bullous palmoplantar eczema, asthma such as bronchial asthma, bronchial asthma, autoimmune asthma, conditions with T cell infiltration and conditions with chronic inflammatory responses, immune responses to foreign antigens such as fetal ABO blood group during pregnancy, chronic pulmonary inflammatory diseases, autoimmune myocarditis, leukocyte adhesion deficiency, lupus including lupus nephritis, cerebral lupus, childhood lupus, non-renal lupus, extrarenal lupus, discoid lupus and discoid lupus erythematosus, alopecia lupus, systemic lupus erythematosus (SLE) such as cutaneous SLE or subacute cutaneous SLE, neonatal lupus syndrome (NLE) and disseminated lupus, juvenile-onset (Type 1) diabetes including childhood insulin-dependent diabetes mellitus (IDDM), adult-onset diabetes mellitus (Type 2 diabetes) and autoimmune diabetes. Immune responses associated with acute and delayed hypersensitivity mediated by cytokines and T lymphocytes, sarcoidosis, granulomatous diseases including lymphomatoid granulomatosis, Wegener's granulomatosis, agranulocytosis, vasculitis including vasculitis, large-vessel vasculitis (including polymyalgia rheumatica and Gian T-cell (Takayasu) arteritis), medium-sized vasculitis (including Kawasaki disease and polyarteritis nodosa / periarteritis nodosa), microscopic polyangiitis, immune vasculitis, CNS vasculitis, cutaneous vasculitis, hypersensitivity vasculitis, necrotizing vasculitis and ANCA-associated vasculitis, for example, systemic necrotizing vasculitis.Also contemplated are Churg-Strauss vasculitis or syndrome (CSS) and ANCA-associated small-vessel vasculitis, temporal arteritis, aplastic anemia, autoimmune aplastic anemia, Coombs-positive anemia, Diamondfan anemia, and Blackfan anemia. Hemolytic anemia or immune-mediated hemolytic anemia includes autoimmune hemolytic anemia (AIHA), Addison's disease, autoimmune neutropenia, pancytopenia, leukopenia, diseases associated with leukodialysis, CNS inflammatory disorders, Alzheimer's disease, Parkinson's disease, multiple organ injury syndromes such as those secondary to sepsis, trauma, or Hemorrhage, antigen-antibody complex-mediated disorders, antiglomerular basement membrane disease, antiphospholipid syndrome, allergic neuritis, Behçet's disease / syndrome, Castleman syndrome, Goodpasture's syndrome, Bow syndrome, Sjögren's syndrome, Stevens-Johnson syndrome, pemphigoid, pemphigoid such as cutaneous pemphigoid, pemphigus (including pemphigus vulgaris, pemphigus foliaceus, mucous membrane pemphigus, and pemphigus erythematosus), autoimmune polyendocrine deficiency, Reiter's disease or syndrome, burns, preeclampsia, immune complex disorders such as immune complex nephritis, antibody-mediated nephritis autoimmune or immune-mediated thrombocytopenias such as idiopathic thrombocytopenic purpura (ITP), including chronic or acute ITP; scleritis, such as idiopathic dermatoscleritis; episcleritis; autoimmune diseases of the testes and ovaries, including autoimmune orchitis and oophoritis; autoimmune endocrine disorders, including primary hypothyroidism, hypoparathyroidism, thyroiditis; autoimmune thyroiditis, Hashimoto's disease, chronic thyroiditis (Hashimoto's thyroiditis), or subacute thyroiditis; autoimmune thyroid disease disease, idiopathic hypothyroidism, Graves' disease, polyglandular syndromes, e.g. autoimmune polyglandular syndrome (or polyendocrinopathy syndrome), paraneoplastic syndromes, neurological paraneoplastic syndromes, e.g. Lambert-Eaton myasthenic syndrome or Eaton-Lambert syndrome, stiff-man syndrome or stiff-person syndrome, encephalomyelitis, e.g. allergic encephalomyelitis or allergic encephalomyelitis and experimental allergic encephalomyelitis (EAE), experimental autoimmune encephalomyelitis, myasthenia gravis, e.g. thymoma-associated myasthenia gravis, cerebellar degeneration, neuromyotonia,Opsoclonus or opsoclonus-myoclonus syndrome (OMS) and sensory neuropathy, multifocal motor neuropathy, Sheehan's syndrome, autoimmune hepatitis, chronic hepatitis, lupoid hepatitis, t-cell hepatitis, chronic active hepatitis or autoimmune chronic active hepatitis, lymphocytic interstitial pneumonia (LIP), bronchiolitis obliterans (non-transplant) vs. NSIP, Guillain-Barré syndrome, Berger's disease (IgA nephropathy), linear IgA dermatosis, acute febrile neutrophilic dermatosis, subcorneal dermatosis, transient acantholytic dermatosis, cirrhosis, e.g. primary biliary cirrhosis and cirrhosis, autoimmune enteropathy syndrome, celiac disease or celiac disease, celiac sprue (gluten enteropathy) , refractory sprue, idiopathic sprue, cryoglobulinemia, amyotrophic lateral sclerosis (ALS; Lou Gehrig's disease), coronary artery disease, autoimmune inner ear diseases such as autoimmune inner ear disease (AIED), autoimmune hearing loss, polychondritis such as refractory or relapsing or relapsing polychondritis, pulmonary alveolar proteinosis, Cogan's syndrome / non-syphilitic interstitial keratitis, Bell's palsy, Sweet's disease / syndrome, autoimmune rosacea, shingles-associated pain, amyloidosis, non-cancerous lymphocytosis, primary lymphocytosis (including monoclonal B-cell lymphocytosis (benign monoclonal gammopathy and monoclonal gammopathy of undetermined significance, MGUS)), Peripheral neuropathy, paraneoplastic syndromes, central nervous system channelopathies such as epilepsy, migraine, cardiac arrhythmias, myopathy, hearing loss, blindness, periodic paralysis and channelopathies, autism, inflammatory myopathies, focal or segmental glomerulosclerosis (FSGS), endocrine ophthalmopathy, uveoretinitis, chorioretinitis, autoimmune liver disease, fibromyalgia, multiple endocrine disorders, Schmidt's syndrome, adrenal inflammation, gastrotrophy, senile dementia, demyelinating diseases such as autoimmune demyelinating diseases and chronic inflammatory demyelinating polyneuropathy, Dressler's syndrome, green alopecia, alopecia totalis, crest syndrome (calcification, leukoencephalopathy), Inno phenomenon, esophageal dysmotility, sclerodatal) and telangiectasia), male and female autoimmune infertility, e.g., due to antisperm antibodies, mixed connective tissue disease, Chagas disease, recurrent miscarriage, farmer's lung, erythema multiforme, post-cardiotomy syndrome, Cushing's syndrome, bird fancier's lung, allergic granulomatous vasculitis, benign lymphocytic vasculitis, Alport syndrome, alveolitis such as allergic alveolitis and fibrosing alveolitis, interstitial lung disease, transfusion reactions, leprosy, malaria, parasitic diseases such as leishmaniasis, cypanosomiasis, schistosomiasis, ascariasis, aspergillosis, Sumpter ulcers syndrome, Kaplan's syndrome, dengue fever, endocarditis, endocardial fibrosis, endomyocardial fibrosis, diffuse interstitial pulmonary fibrosis, interstitial pulmonary fibrosis, pulmonary fibrosis, idiopathic pulmonary fibrosis, cystic fibrosis, endophthalmitis, erythrocytosis, fetal erythrocytosis, eosinophilia, Shulman's syndrome, iron fibrosis, cyclitis such as cyclitis, heterochronic cyclitis, iridocyclitis (acute or chronic), or Hoof-cyclitis, Henoch-Schönlein purpura, human immunodeficiency virus (HIV) infection, SCID, acquired immune deficiency syndrome (AIDS), echovirus infection, sepsis, endotoxemia, pancreatitis, thyrotoxicosis, parvovirus, pulmonary fibrosis ... rubella infection, rubella virus infection, post-vaccination syndrome, congenital rubella infection, Epstein-Barr virus infection, mumps, Evan's syndrome, autoimmune gonadal failure, Sydenham's chorea, poststreptococcal nephritis, tubulitis, thyrotoxicosis, spinal cord disease, choroiditis, Gian T-cell polymyalgia, chronic hypersensitivity pneumonitis, keratoconjunctivitis sicca, keratoconjunctivitis, idiopathic epidemic nephritis, minimal change nephropathy, benign immune and familial injuries, autoimmune reperfusion organ, retinal transplant, joint inflammation, bronchitis, chronic obstructive airway / pulmonary disease, silicosis, aphthous stomatitis, atherosclerotic disorders, asperniogenes, autoimmune hemolysis,Boeck's disease, cryoglobulinemia, Dupuytren's contracture, intracapsular capsulitis, allergic enterocolitis, erythema nodosum liporum, idiopathic facial paralysis, chronic fatigue syndrome, rheumatic fever, Hammann-Rich disease, sensorineural hearing loss, hemoglobinuria, paroxysmal hemoglobinuria, hypogonadism, focal ileitis, leukopenia, infectious mononucleosis, transverse myelitis, primary idiopathic myxedema, nephropathy, symptomatic ophthalmia, granulomatous orchitis, pancreatitis, acute polyradiculitis, pyoderma gangrenosum, Quervain's thyroiditis, acquired spinal cord atrophy, nonmalignant thymoma, vitiligo, toxic shock syndrome, food poisoning, and inflammatory bowel disease associated with T-cell infiltration. Cavity, leukocyte adhesion defects, immune responses related to cytokine- and T-lymphocyte-mediated acute and delayed hypersensitivity, diseases with leukocytosis, multiorgan injury syndrome, antigen-antibody-mediated diseases, anti-glomerular basement membrane disease, allergic neuritis, autoimmune polyendocrine deficiency, oophoritis, primary myxedema, autoimmune atrophic gastritis, sympathetic ophthalmia, rheumatic diseases, mixed connective tissue disease, renal syndrome, insulitis, polyendocrine deficiency, autoimmune polyglandular syndrome type I, adult-onset idiopathic hypoparathyroidism (AOIH), cardiomyopathies such as dilated cardiomyopathy, epidermolysis bullosa acquisita (EBA), hemochromatosis eosinophilia, myocarditis, renal syndrome, primary sclerosing cholangitis, suppurative or non-suppurative sinusitis, acute or chronic sinusitis, etomoid, frontal, maxillary, or sphenoiditis, eosinophilia, pulmonary eosinophilia, eosinophilic myalgia syndrome, Löffler's syndrome, chronic eosinophilic pneumonia, eosinophil-related diseases such as tropical eosinophilia, allergic bronchopulmonary aspergillosis, aspergilloma, or eosinophil-containing granuloma, anaphylaxis, seronegative spondyloarthritis, polyendocrine autoimmune disease, sclerosing cholangitis, scleral, episcleral, chronic mucocutaneous candidiasis, Bruton's syndrome, transient infantile hypochondritis Gammopathy, Wiskott-Aldrich syndrome, ataxia-telangiectasia syndrome, vascular ectasia, autoimmune disorders associated with collagen diseases, rheumatism, neurological diseases, lymphadenitis, decreased blood pressure response, vascular dysfunction, tissue injury, cardiovascular ischemia, hyperalgesia, renal ischemia, cerebral ischemia, and diseases involving angiogenesis, allergic hypersensitivity, glomerulonephritis, injury, ischemia-reperfusion injury, reperfusion injury of the myocardium or other tissues, lymphocytic cholangitis, inflammatory dermatoses, skin diseases with an acute inflammatory component, multiple organ failure, bullous renal necrosis, acute suppurative meningitis or other central nervous system inflammatory disorders,Ocular and orbital inflammatory disorders, granulocyte transfusion-associated syndrome, cytokine-induced toxicity, narcolepsy, acute severe inflammation, chronic refractory inflammation, pyelitis, intra-arterial hyperplasia, peptic ulcer, valvulitis, graft-versus-host disease, contact hypersensitivity, asthmatic airway hyperresponsiveness, and endometriosis are also considered.

[0192] Further embodiments relate to the treatment or prevention of microbial infections and / or the use of microbial antigens. The microbial infection or antigen to be treated or prevented can be an antigen associated with any microbial infection known in the art, or, for example, anthrax, cervical cancer (human papillomavirus), diphtheria, hepatitis A, hepatitis B, Haemophilus influenzae type b (Hib), human papillomavirus (HPV), influenza (Flu), Japanese encephalitis (JE), Lyme disease, measles, meningococcus, monkeypox, mumps, whooping cough, pneumococcus, polio, rabies, rotavirus, rubella, shingles (herpes zoster), smallpox, tetanus, typhoid fever, tuberculosis (TB), chickenpox (varicella), and yellow fever.

[0193] In some embodiments, the methods and compositions may be for vaccinating an individual to prevent a medical condition such as cancer, inflammation, infection, and the like. [Example]

[0194] Example

[0195] The following examples are included to demonstrate preferred embodiments of the invention. It should be understood by those of skill in the art that the techniques disclosed in the examples below represent techniques discovered by the inventors to function well in the practice of the invention, and can therefore be considered to constitute preferred modes for its practice. However, those of skill in the art should, in light of the present disclosure, understand that many changes can be made in the specific embodiments disclosed and still obtain like or similar results without departing from the spirit and scope of the invention.

[0196] Detailed description of PSC manipulation target cell culture method Stage 0: Generation of a PSC master cell bank PSC lines are cultured in an appropriate cell culture vessel (e.g., in Matrigel or on a laminin-coated plate) containing serum-free PSC culture medium (basal medium supplemented with a cytokine cocktail including bFGF, TGFβ, FLT3L, Noggin, Activin B, Biotin, LIF, and others) for 12-72 hours, followed by the addition of the TCR gene delivery vector and further culture for 1-7 days. After validation (and sorting and single cloning, if necessary), TCR-engineered PSC lines can be used to establish a master cell bank that can be cryopreserved for storage and / or maintained in cell culture via passaging. Stage 1: Differentiation of PSCs to HSPCs TCR-engineered PSC cells are gently dissociated into single cells (e.g., via cell dissociation reagents such as Accutase, Versene, or TrypLE) and then transferred to an appropriate cell culture vessel (e.g., ultra-low attachment plates or AggreWell) in HSC differentiation medium A (basal medium containing GlutaMAX, L-ascorbic acid, monothioglycerol, insulin-transferrin-selenium, activin A, BMP-4, bFGF, VEGF, SB431542, CHIR99021, and others) supplemented with ROCK inhibitor for 12-48 h to form embryonic bodies (EBs) or monolayers. The medium is then replaced with fresh HSC differentiation medium A for the following 24-72 h. On days 3-5, EBs or monolayers are collected and replated in appropriate cell culture vessels (e.g., ultra-low attachment plates or untreated tissue culture plates) in HSC differentiation medium B (basal medium containing GlutaMAX, L-ascorbic acid, monothioglycerol, insulin-transferrin-selenium, heparin, BMP-4, bFGF, VEGF, SCF, TPO, Flt3 ligand, IL-3, IL-6, IL-11, IGF-1, IGF-2, SB431542, EPO, and others) for 6-12 days. HSC differentiation medium B is refreshed every other day. At the end of the culture, CD34 cells in suspension are plated. + HSCs are harvested and cryopreserved. Stage 2: Differentiation of HSCs into target cells TCR-engineered PSC-derived HSCs are then differentiated into TARGET cells in differentiation medium for 4–10 weeks without feeders. Non-tissue culture-treated plates are coated with TARGET Culture Coating (TARGETc) material (DLL-1 / 4, VCAM-1 / 5, retronectin, and others). CD34 + HSCs are suspended in TARGET Expansion (TARGETe) medium (a basal medium containing serum albumin, recombinant human insulin, human transferrin, 2-mercaptoethanol, L-ascorbic acid, IL-7, SCF, TPO, IL-3, IL-6, Flt3 ligand, SDF-1α, human LDL, UM171, SB203580, and additives), seeded into coated wells of plates, and cultured for 12–14 days. TARGETe medium is refreshed every 3–4 days. The cells are then collected, suspended in TARGET Maturation (TARGETm) medium (a basal medium containing serum albumin, recombinant human insulin, human transferrin, 2-mercaptoethanol, L-ascorbic acid, IL-7, SCF, IL-6, IL-15, Flt3 ligand, SDF-1α, human LDL, UM171, SB203580, and additives), seeded into coated wells of plates, and cultured for an additional 14–28 days. The TARGETm medium is refreshed every 3–4 days. Stage 3: Target cell proliferation The differentiated TARGET cells are stimulated with TCR cognate antigens (e.g., proteins, peptides, lipids, phosphoantigens, small molecules, etc.) or nonspecific TCR stimulatory reagents (e.g., anti-CD3 / anti-CD28 antibodies or antibody-coated beads, concanavalin A, PMA / ionomycin, etc.) with or without antigen-presenting cells (e.g., irradiated healthy donor PBMCs, artificial APCs, etc.) and grown in T cell culture medium for up to one month. Cell culture basal media include, but are not limited to, CTS OpTmizer, TexMACS, RPMI, DMEM, and X-Vivo15. Cell culture approaches can be serum-free and feeder-free. Cultures may be supplemented with T cell supporting cytokines (e.g., IL-2, IL-4, IL-7, IL-12, IL-15, IL-18, IL-21, TNFα, SDF-1α, TGF-β, and others), small molecules, and additives (e.g., Wnt activators or glycogen synthase kinase-3 (GSK-3) inhibitors such as Wnt3A, CHIR99021, AR-A014418, TWS119, LY2090314, 9-ING-41, lithium chloride (LiCl), BIO(6-bromoindirubin-3-oxime, 6-bromoindirubin-3′-oxime, or tyrosine kinase inhibitors such as dasatinib, ibrutinib, acalabrutinib, or zanubrutinib). TARGET cell derivative In some embodiments, PSC master cell lines / banks and their derived HSCs and TARGET cells can be further engineered to express additional transgenes. In one embodiment, such transgenes encode disease-targeting molecules such as chimeric antigen receptors (CARs), T cell receptors (TCRs), and other natural or synthetic receptors / ligands. In another embodiment, such transgenes can encode IL-2, IL-4, IL-7, IL-10, IL-12, IL-15, IL-18, IFN-γ, TNF-α, TL1A, CD27, CD28, 4-1BB, OX40, ICOS, DAP10, and other T cell regulatory proteins. In another embodiment, such transgenes can encode Bcl11b, Batf3, ThPOK, FOXP3, Runx3, and other transcription factors. Transgenes can be introduced into expanded TARGET cells or their progenitors (HSCs, newly differentiated TARGET cells, and expanding TARGET cells) at various culture stages. In some embodiments, PSC master cell lines / banks and their derived HSCs and TARGET cells can be further engineered to disrupt selected genes using gene editing tools (CRISPR, TALEN, Zinc-Finger, and others). In one embodiment, the disrupted gene encodes a T cell immune checkpoint inhibitor (PD-1, CTLA-4, TIM-3, LAG-3, CD161, and others). In another embodiment, the disrupted gene encodes a T cell regulatory protein (e.g., TET2, PI3Kδ / γ, DGK, DNMT3a, Suv39h1, and others). Deletion of these negative regulatory genes can enhance the ability of TARGET cells to fight disease and create resistance to disease-induced anergy and tolerance. In some embodiments, PSC master cell lines / banks and their derived HSCs, as well as TARGET cells or expanded TARGET cells, may be further engineered to render them suitable for allogeneic adoptive transfer, thereby making them suitable for serving as ready-to-use cell products. In one embodiment, genes encoding MHC molecules or MHC expression / display regulatory molecules (MHC molecules, B2M, CIITA (class II transcription activator that controls the induction of MHC class II mRNA expression), and others) are engineered. The lack of MHC molecule expression on TARGET cells renders them resistant to allogeneic host T cell-mediated depletion. In another embodiment, MHC class I-deficient TARGET cells are further engineered to overexpress HLA-C, HLA-E, HLA-G, and CD47 genes, which confer resistance to host NK cell-mediated depletion. Genetically engineered PSC master cell lines / banks can be cryopreserved for storage or maintained in culture through passaging. PSC-derived HSCs can be used fresh or cryopreserved for future use. TARGET cell products and derivatives can also be used fresh or cryopreserved for further use. Furthermore, various intermediate cell products generated during PSC-to-TARGET cell culture can be suspended, stored for cryopreservation, and harvested for continued production. Genetically engineered primary human CD34 + Compared with our previous invention for the in vitro generation of target cells by culturing HSCs, this invention provides an in vitro differentiation method that can generate target cells from genetically engineered PSCs as an unlimited resource. This new method significantly improves the process for scale-up production and GMP-compliant manufacturing of therapeutic cells for human use. The cell product, TARGET cells, can be derived from their natural counterpart T cells as well as their counterpart T cells or other starting cells (e.g., primary human CD34 T cells) generated using other ex vivo culture methods (e.g., ATO culture methods). +These PSC-derived TARGET cells display a distinct phenotype / functionality from that of their T cell counterparts derived from PSCs (HSCs), making these PSC-derived TARGET cells a unique cell product. Unique features of PSC-derived TARGET cell differentiation cultures include: 1) It is ex vivo and feeder-free. 2) High CD34+ purity eliminates the need to purify and select CD34+ HSCs after differentiation of PSCs into HSCs. 3) Robust endogenous TCR recombination (γδTCR and conventional αβTCR) occurs during T cell differentiation without transgenic TCR manipulation or the use of T-iPSCs, demonstrating that our culture platform resembles natural T cell development. 4) It can generate human CD8 single-positive T cells and CD4 single-positive T cells, which can be further differentiated into T helper 2 (Th2) T cells. 5) TARGET cells possess monoclonal innate TCRs, so there is no risk of GvHD. 6) It supports the synchronous differentiation of transgenic TARGET cells, thereby eliminating the presence of undifferentiated progenitor cells and other lineages of bystander immune cells. 7) The resulting TARGET cell product contains a homogenous and pure population of monoclonal TCR-armed T cells. There are no escaped random T cells, no other lineages of immune cells, and no undifferentiated progenitor cells. Therefore, no purification steps are required. 8) an unlimited supply of source PSCs and product TARGET cells; and 9) Unique phenotype of TARGET cells - monoclonal TCR+ random αβ TCR- CD3+. As disclosed herein, proof-of-principle studies have been performed to demonstrate the efficacy of iNKT TCR-engineered TARGET cells derived from PSCs ( PSC iNKT cells) and gamma-delta TCR engineered TARGET cells ( PSC This demonstrates the successful generation of BCMA CAR ( PSCBCAR-iNKT cell products) PSC Further manipulation of iNKT cells also proved successful. Pilot CMC, pharmacology, efficacy, and safety studies were conducted analyzing these cell products. "Off-the-shelf PSC-derived BCMA-targeted CAR-armed invariant natural killer T ( PSC Generation and characterization of BCAR-iNKT cells Invariant natural killer T (iNKT) cells are a small subpopulation of αβ T lymphocytes that have the ability to bridge innate and adaptive immunity. Unlike conventional αβ T cells, the T cell receptor (TCR) of iNKT cells recognizes lipid antigens presented by the major histocompatibility complex (MHC)-like molecule CD1d rather than MHC itself. Due to this unique property, iNKT cells do not cause graft-versus-host disease (GvHD) when allogeneically transplanted. Furthermore, iNKT cells possess several other unique features that make them ideal cell carriers for developing off-the-shelf cell therapies for cancer. 1) They have a role in cancer immunosurveillance. 2) They have a remarkable ability to target tumors independently of tumor antigen and major histocompatibility complex (MHC) restriction. 3) They can use multiple mechanisms to attack tumor cells through direct killing and adjuvant effects. However, the development of allogeneic, off-the-shelf iNKT cell products is largely hindered by their availability—these cells are extremely rare in humans and highly variable (approximately 0.001–1% in human blood), making it extremely difficult to produce therapeutic numbers of iNKT cells from allogeneic human donor blood cells. Two conventional methods have been used to generate sufficient iNKT cells for therapeutic use. One method involves screening a large number of donors to identify "super donors" who naturally possess a high percentage of iNKT cells in their peripheral blood. iNKT cells are enriched by a magnetic bead-based purification procedure and then expanded by either anti-CD3 / CD28 bead stimulation or coculture with alpha-galactosylceramide (αGC)-loaded antigen-presenting cells. Although expansion can be achieved by this method, the expansion fold is limited and unreliable. Another method is based on the artificial thymic organoid (ATO) culture system, which supports the in vitro differentiation of human hematopoietic stem cells (HSCs) into iNKT cells after genetic modification of HSCs with iNKT TCR. While this method can generate iNKT cells at a high yield, production requires the use of feeder cells of murine origin, which poses a significant challenge for developing a reliable process for GMP-compliant manufacturing. Therefore, novel methods that can reliably generate large amounts of homogenous monoclonal populations of iNKT cells using feeder-free differentiation systems are crucial for developing off-the-shelf iNKT cell therapies. CMC research- PSC BCAR-iNKT cells (Figures 16 and 17) PSCs were transduced with a Lenti / iNKT-BCAR-(GFP) vector encoding a human iNKT TCR gene, a BCMA-targeting CAR gene, and an optional GFP reporter gene to establish a genetically engineered PSC master cell line / bank, which was then placed into feeder-free / serum-free ex vivo PSC-derived CAR-iNKT cell culture to generate PSC-derived BCMA CAR-armed iNKT ( PSC We generated BCAR-iNKT (iNKT) cells. Both embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) can be used as starting PSC source cells. Data generated by using an iPSC cell line (DMD; Figure 16) or an ES cell line (H1; Figure 17) are presented. Through a three-stage culture (HSC differentiation, iNKT differentiation, iNKT expansion) over approximately 5-6 weeks, pure clones were obtained. PSCNote that BCAR-iNKT cells were successfully generated. Pharmacological research PSC BCAR-iNKT cells (Figure 18) PSC The phenotype and functionality of BCAR-iNKT cells were investigated using flow cytometry (Figure 18). PSC BCAR-iNKT cells exhibit similar phenotype and functionality to iPSC-derived PSC Data are presented for BCAR-iNKT cells. Conventional αβ T cells derived from healthy donor PBMCs engineered to express the same BCAR were included as a benchmark control (denoted BCAR-T cells). PSC BCAR-iNKT cells displayed typical human iNKT cell phenotype and functionality. They expressed CD4 and CD8 co-receptors with a mixed pattern (CD4 / CD8 double negative and CD8 single positive). They also expressed T cell activation markers (CD69 hi and CD62L lo ) and produced very high levels of effector molecules (e.g., perforin and granzyme B). Notably, similar ex vivo generated primary human CD34 + HSC-derived human iNKT cells, PSC BCAR-iNKT cells express significantly low levels of surface HLA-I molecules and nearly undetectable surface HLA-II molecules, suggesting that these cells may be suitable for "off-the-shelf" cell therapy because they may resist allorejection mediated by host T cells when adoptively transferred into an allogeneic host. In vitro efficacy and MOA studies - PSC iNKT cells (Figures 19 and 20) even if they are not engineered to express additional tumor-targeting molecules such as chimeric antigen receptors (CARs). PSCiNKT cells are already capable of targeting tumor cells via NK receptor-mediated pathways. We established an in vitro tumor cell killing assay to study their tumor-killing ability (Figure 19A). Conventional αβ T cells derived from healthy donor PBMCs that were not engineered to express CARs were included as a control (denoted as PBMC-T cells). Various human tumor cell lines were engineered to overexpress a firefly luciferase and enhanced green fluorescent protein dual reporter (denoted as FG), which facilitates detection of tumor cell killing using highly sensitive luciferase activity assays and flow cytometry assays. Four FG reporter-marked human tumor cell lines were used in this study, including the human melanoma cell line A375-FG, the human breast cancer cell line MDA-FG, the human ovarian cancer cell line OVCAR8-FG, and the prostate cancer cell line PC3-FG. PSC iNKT cells, unlike control PBMC-T cells, effectively killed all four types of human tumor cells (Figures 19B-19E). Blockade of NK activating receptors (e.g., DNAM-1) PSC The tumor cell killing effect of iNKT cells was reduced, confirming their NK-activating receptor-mediated tumor targeting function (Figures 20A and 20B). This mechanism of action (MOA) was supported by the detection of corresponding NK-activating receptor ligands (e.g., DNAM-1 ligands such as Nectin-2 and PVR) on all four human tumor cell lines tested. PSC Targeting a wide range of human tumors via this intrinsic NK function of iNKT cells PSC This suggests the potential of iNKT cells (Fig. 20C). In vitro efficacy and MOA studies - PSC BCAR-iNKT cells (Figure 21) We established an in vitro tumor cell killing assay for this study (Figures 21A and 21C). BCMA-targeted CAR-armed PSC-derived human iNKT ( PSC BCAR-iNKT) cells have been investigated as potential therapeutic candidates. PSCiNKT), as well as conventional αβT cells derived from PBMCs of healthy donors (PBMC-T) and those engineered to express the same BCMA-targeting CAR (BCAR-T cells) were included as control effector cells. Two human tumor cell lines were used in this study: 1) BCMA + CD1d - 1) a human MM cell line, MM.1S, which served as a target for CAR-mediated killing (Figure 21A); and 2) an MM.1S cell line engineered to overexpress human CD1d, MM.1S-CD1d (which expresses BCMA + CD1d + Both human tumor cell lines were engineered to express firefly luciferase (Fluc) and enhanced green fluorescent protein (EGFP) dual reporters. Expression of human CD1d enabled tumor cells to present iNKT TCR-cognate glycolipid antigens, such as endogenous tumor lipid antigens or synthetic lipid antigens like αGC, sensitizing CD1d tumor cells to the iNKT TCR / CD1d / glycoantigen-mediated tumor killing pathway. Expression of Fluc and EGFP facilitated detection of tumor cell killing using highly sensitive luciferase activity and flow cytometry assays. The resulting MM.1S-FG and MM.1S-CD1d-FG human multiple myeloma cell lines were then utilized in the study. PSC BCAR-iNKT cells effectively killed MM.1S-FG tumor cells with a potency comparable to that of BCAR-T cells (Figure 21B). PSC iNKT cells, unlike non-CAR-armed PBMC-T cells, demonstrated robust efficacy in killing MM.1S-FG tumor cells, likely via the NK killing pathway (Figure 21B). PSC BCAR-iNKT cells effectively killed MM.1S-hCD1d-FG tumor cells with a potency comparable to that of conventional BCAR-T cells (Figure 21D). Importantly, in the presence of the cognate lipid antigen (αGC), BCAR-iNKT cells, but not BCAR-T cells, PSCBCAR-iNKT cells exhibited enhanced tumor killing efficacy, likely due to additional activation of the TCR / CD1d / αGC tumor killing pathway (FIG. 21D). In summary, these results are PSC We demonstrate that CAR-iNKT cells can target tumors using three mechanisms: 1) the CAR-dependent pathway, 2) the iNKT TCR-dependent pathway, and 3) the NK pathway. PSC This unique triple targeting ability of CAR-iNKT cells is attractive because it could potentially circumvent antigen escape, a phenomenon reported in autologous CAR-T treatment clinical trials in which tumor cells downregulated the expression of CAR-targeted antigens to avoid attack from CAR-T cells. Safety and immunogenicity studies PSC BCAR-iNKT cells (Figure 22) There are two safety and immunogenicity concerns with allogeneic cell therapy: a) GvHD response, and b) host-versus-graft (HvG) response. PSC The possible GvHD and HvG risks for BCAR-iNKT cell products were examined (Figure 22). PSC iNKT cells were studied and showed similar results (data not shown). GvHD is a major safety concern. However, iNKT cells are not expected to induce GvHD because they do not respond to mismatched HLA molecules and protein autoantigens. 1 This concept is evidenced by the lack of GvHD in human clinical experience with allogeneic HSC transfer and autologous iNKT transfer. 2、3 , supported by our in vitro mixed lymphocyte reaction (MLR) assay designed to study the GvH response (Figure 22A). PSC Note that BCAR-iNKT cells did not respond to random allogeneic healthy donor PBMCs, in sharp contrast to that of conventional PBMC-T cells (Figure 22B). On the other hand, HvG risk is an efficacy concern mediated primarily through the elimination of allogeneic therapeutic cells by host immune cells, primarily by conventional CD8 and CD4 T cells that recognize mismatched HLA-I and HLA-II molecules. In vitro mixed lymphocyte reaction (MLR) assays designed to study HvG responses demonstrated significantly higher levels of HvG responses compared with conventional BCAR-T cells. PSC BCAR-iNKT cells induced an overall reduced HvG response when cultured with random allogeneic healthy donor PBMCs (Figures 22C and 22D), likely due to the expression of surface HLA-I molecules and nearly undetectable HLA-II molecules compared to those of conventional BCAR-T cells (Figures 22E and 22F). In summary, these results are PSC This strongly supports CAR-iNKT cells as an ideal candidate for an off-the-shelf cell therapy that is GvHD-free and HvG-resistant. "Off-the-shelf PSC-derived gamma delta T ( PSC Generation and characterization of γδT) cells Gamma delta T (γδ T) cells are a small subpopulation of T lymphocytes that have the ability to bridge innate and adaptive immunity. The majority of γδ T cells in adult blood display the Vγ9Vδ2 TCR and respond to small phosphorylated non-peptide antigens, called phosphoantigens (pAgs), that are commonly produced by malignant cells. 5Unlike conventional αβ T cells, γδ T cells do not recognize polymorphic classical major histocompatibility complex (MHC) molecules and therefore pose no risk of GvHD when adoptively transferred into allogeneic hosts. Furthermore, γδ T cells possess several other unique characteristics that make them ideal cell carriers for developing off-the-shelf cell therapies for cancer: 1) they have a role in cancer immunosurveillance; 2) they have a remarkable ability to target tumors independently of tumor antigen and major histocompatibility complex (MHC) restriction; 3) they can use multiple mechanisms to attack tumor cells through direct killing and adjuvant effects; and 4) they express the surface receptor FcγRIII (CD16), which participates in antibody-dependent cellular cytotoxicity (ADCC) and can potentially be combined with monoclonal antibodies for cancer therapy. However, the development of allogeneic, off-the-shelf γδ T cell products is largely hindered by their availability—these cells are extremely rare and highly variable in humans (approximately 1–5% T cells in human blood), making it extremely difficult to produce therapeutic numbers of γδ T cells from allogeneic human donor blood cells. Current methods for generating γδ T cells, particularly the Vγ9Vδ2 subset, for adoptive therapy involve in vitro or in vivo expansion of peripheral blood mononuclear cells (PBMCs)-derived γδ T cells using aminobisphosphonates such as zoledronate (ZOL). However, this method produces highly variable yields of γδ T cells depending on the PBMC donor. Most importantly, such γδ T cell products are likely to contain bystander αβ T cells, thereby incurring a risk of GvHD. Therefore, novel methods that can reliably generate large quantities of homogenous monoclonal populations of γδ T cells using a feeder-free differentiation system are crucial for developing off-the-shelf γδ T cell therapies. CMC research- PSC γδT cells (Figure 23) PSCs were transduced with a Lenti / γδT vector encoding a pair of human γδT Vγ9 and Vδ2 TCR genes to establish a genetically engineered PSC master cell line / bank, which were then placed into feeder-free / serum-free ex vivo PSC-derived γδT cell cultures to generate PSC-derived γδT ( PSC We generated γδT (γδT) cells. Both embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) can be used as starting PSC source cells. Data generated using the H1 ES cell line (Figure 23) are presented. Through a three-stage culture (HSC differentiation, γδT differentiation, γδT expansion) over approximately 5-6 weeks, pure clones were obtained. PSC Note that γδ T cells were successfully generated. Pharmacological research PSC γδT cells (Figure 24) PSC The phenotype and functionality of γδ T cells were investigated using flow cytometry (FIG. 24). H1 ESC-derived PSC Data for γδ T cells are presented. Conventional αβ T cells and γδ T cells derived from healthy donor PBMCs were included as staining controls (denoted as PBMC-T and PBMC-γδ T cells, respectively). PSC The γδ T cells displayed typical human γδ T cell phenotype and functionality. They expressed T cell activation markers (e.g., CD69 hi CD62L lo ) and inflammatory homing markers (e.g., CXCR3 hi CCR5 hi ) and produced very high levels of effector molecules (e.g., cytotoxic molecules such as perforin and granzyme B). PSC γδ T cells express a broad range of NK activating receptors (e.g., NKG2D, DNAM-1, NKp30, NKp44) at levels much higher than natural conventional PBMC-T cells and even higher than natural PBMC-γδ T cells, which may be advantageous for their cancer therapeutic application. PSC This suggests a potent NK function of γδ T cells. In vitro efficacy and MOA studies -PSC γδT cells (Figure 25) We established an in vitro tumor cell killing assay for this study (Figures 25A and 25C). PSC γδT) cells were investigated as potential therapeutic candidates. Conventional αβT (PBMC-T) cells derived from healthy donor PBMCs were included as a control. Two human tumor cell lines were used in this study: 1) the human melanoma cell line A375 (Figure 25A); 2) the human multiple myeloma cell line MM.1S (Figure 25C). Both human tumor cell lines were engineered to express a dual reporter of firefly luciferase and enhanced green fluorescent protein (FG). Expression of Fluc and EGFP facilitates detection of tumor cell death using highly sensitive luciferase activity assays and flow cytometry assays. The resulting A375-FG and MM.1S-FG human tumor cell lines were then utilized in the study. PSC γδ T cells effectively kill both A375-FG and MM.1S-FG tumor cells, in contrast to PBMC-T cells, which did not exhibit tumor cell killing, and target a broad range of human tumor cells independently of the expression of additional tumor-targeting molecules (e.g., CARs). PSC These CAR-independent tumor cell targeting mechanisms demonstrated the unique capabilities of γδ T cells (Figures 24B and 24D). PSC This may be mediated via NK and / or γδ TCR pathways, as supported by the high levels of NK activating receptor expression on γδ T cells (FIG. 24) and enhanced tumor cell killing in the presence of the γ9δ2 TCR stimulator zoledronate (ZOL) (FIG. 25D). PSC The multiple tumor-targeting mechanisms of γδ T cells are attractive because they could potentially circumvent antigen escape, a phenomenon reported in autologous CAR-T treatment clinical trials, in which tumor cells downregulated the expression of CAR-targeted antigens to avoid attack by CAR-T cells. Safety and immunogenicity studies PSC γδT cells (Figure 26) There are two immunogenic concerns for allogeneic cell therapy: a) graft-versus-host (GvH) responses, and b) host-versus-graft (HvG) responses. GvHD is a major safety concern. However, γδ T cells are not expected to induce GvHD because they do not respond to mismatched HLA molecules and protein autoantigens. This concept is evidenced by the lack of GvHD in human clinical experience with allogeneic HSC transfer and autologous γδ T cell transfer, and is supported by our in vitro mixed lymphocyte reaction (MLR) assay (Figure 26A). PBMC-γδ T cells also PSC Note that γδ T cells also failed to respond to allogeneic PBMCs, in stark contrast to the response of conventional PBMC-T cells (FIG. 26B). On the other hand, HvG risk is an efficacy concern mediated primarily through the elimination of allogeneic therapeutic cells by host immune cells, primarily by conventional CD8 and CD4 T cells that recognize mismatched HLA-I and HLA-II molecules. Interestingly, compared with PBMC-derived αβ T (PBMC-T) cells, PBMC-γδ T cells expressed reduced levels of HLA-I and HLA-II molecules, but PSC γδT expressed even reduced levels of HLA-I molecules and nearly undetectable HLA-II molecules (Figure 26C). This HLA-I / II expression pattern generally indicates that natural γδT cells are "less immunogenic" than αβT cells, and PSC This suggests that γδT cells exhibit the lowest immunogenicity, which may render these cells resistant to allorejection. Indeed, in an in vitro MLR assay designed to study HvG responses (Figure 26D), PSC γδ T cells induced minimal alloresponses that were significantly lower than those induced by PBMC-T cells as well as PBMC-γδ T cells (FIG. 26E). Taken together, these results suggest that EGFR-10001 is an ideal candidate for an off-the-shelf cell therapy that is GvHD-free and HvG-resistant. PSC Strongly supports γδ T cells. 1 Fujii, S. et al. NKT cells as an ideal anti-tumor immunotherapeutic. Front Immunol 4, 409, doi:10.3389 / fimmu.2013.00409 (2013). 2 Haraguchi, K. et al. Recovery of Valpha24+ NKT cells after hematopoietic stem cell transplantation. Bone Marrow Transplant 34, 595-602, doi:10.1038 / sj.bmt.1704582 (2004). 3 de Lalla, C. et al. Invariant NKT cell reconstitution in pediatric leukemia patients given HLA-haploidentical stem cell transplantation defines distinct CD4+ and CD4- subset dynamics and correlates with remission state. J Immunol 186, 4490-4499, doi:10.4049 / jimmunol.1003748 (2011).

[0197] Although the present disclosure and its advantages have been described in detail, it should be understood that various changes, substitutions, and alterations can be made therein without departing from the spirit and scope of the design, as defined by the appended claims. Moreover, the scope of this application is not intended to be limited to the particular embodiments of the processes, machines, manufacture, compositions of matter, means, methods, and steps described herein. Those skilled in the art will readily understand from the present disclosure that any processes, machines, manufacture, compositions of matter, means, methods, or steps, now existing or later developed, which perform substantially the same function or achieve substantially the same result as the corresponding embodiments described herein, can be utilized in accordance with the present disclosure. Accordingly, the appended claims are intended to include within their scope such processes, machines, manufacture, compositions of matter, means, methods, or steps.

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Claims

1. 1. A method for generating monoclonal TCR-armed genetically engineered T (TARGET) cells using pluripotent stem cells (PSCs), the method comprising transfecting TARGET cells with a selected monoclonal T cell receptor (TCR) gene: (a) as the endogenous TCR gene when T cell reprogrammed induced PSC (T-iPSC) lines are used to generate said TARGET cells; or (b) When a non-T-iPSC PSC line is used to generate the TARGET cells, the exogenous TCR transgene To introduce and differentiating the PSC cells of (a) or (b) to generate the TARGET cells.

2. The monoclonal TCR gene is selected from the group consisting of: Alpha beta TCR, gamma delta TCR, invariant NKT (iNKT) TCR, a non-invariant NKT TCR, and Mucosal-associated invariant T (MAIT) TCR The method of claim 1, wherein the TCR is selected from the group consisting of:

3. The TARGET cell product is Monoclonal TCR positive CD3 positive, HLA-I / II low / negative, Expression of one or more immunomodulatory transgenes, and / or Disrupted expression of one or more endogenous immunoregulatory genes 2. The method of claim 1, comprising a gene expression profile characterized by at least one of:

4. (a) the immune regulatory transgene delivered to the TARGET cell product can encode any one or more of the following: an immune targeting molecule, an immune regulatory molecule, an immune allorejection resistance molecule, a suicide gene, and / or an imaging marker molecule; and (b) the endogenous gene disrupted in the TARGET cells encodes at least one of the following: an immune checkpoint agent selected from PD-1, PD-L1, CTLA-4, LAG-3, TIM-3, TIGIT, B7-H3 / B7-H4, BTLA, VISTA, NKG2A, A2aR, PVRIG, IDO, CD73, CD39, CD96, and CD161, and an immune regulatory molecule; or / and an immune allo-rejection molecule. The method of claim 3.

5. All of the desired genetic modifications intended for a designated TARGET cell product are (a) integrated into a master PSC strain via an All-in-One Engineering (AO-Engineering) strategy; (b) Master PSC lines and their progeny CD34+ hematopoietic stem and progenitor cells (HSPCs) are derived stepwise by an Assembly-Line Engineering (AL-Engineering) strategy; (c) a single transgene or multiple transgenes can be incorporated into the TARGET cell product via a gene delivery vector or a vector-free system; and / or (d) the method of claim 1, wherein a single endogenous gene or multiple endogenous genes of a TARGET cell product are disrupted.

6. PSC has four stages and specific steps: Stage 0, which supports the generation and maintenance of the PSC master cell bank; and Stage 1, which supports the PSC differentiation into CD34+ HSPCs; and Stage 2, which supports the differentiation of the HSPC into mature TARGET cells; and Stage 3, which supports the proliferation of TARGET cells; and an optional "CD4 induction step" that can be added between the Stage 2 and Stage 3 cultures to allow for the generation of CD4 single positive (CD4 SP) TARGET cells; and T Ｈ Another optional "T" antibody can be added in stage 3 cultures to allow for the generation of polarized CD4 SP TARGET cells. Ｈ Polarization step The method of claim 1, wherein the cells are cultured ex vivo to produce TARGET cells.

7. All three culture stages (stages 1, 2, and 3) can be feeder-free and / or serum-free, or the stage 3 culture may comprise feeder cells; and / or The cell culture medium may comprise a basal medium supplemented with one or more factors selected to promote differentiation, proliferation, and sublineage commitment of said PSC-derived TARGET cells; and / or All three stages of ex vivo culture (stages 1, 2, and 3) can achieve high purity, eliminating the need for in-stage purification steps; and / or The PSC-derived HSPCs and / or HSPC-derived TARGET intermediate cell products may be freshly cultured or cryopreserved and then thawed for continued culture; and / or All-in-One Engineered (AO-Engineered) master PSC lines are cultured ex vivo to produce the designated TARGET cell product without the need for additional genetic engineering steps; and / or Assembly-Line Engineered (AL-Engineered) master PSC lines are cultured ex vivo to generate a specified TARGET cell product, requiring additional genetic engineering steps on the PSC-derived HSPCs and / or other TARGET cell precursors. The method of claim 6.

8. The TARGET cell product produced by the methods described herein may be cryopreserved, and / or the cryo-harvested cell product may be stable at room temperature for at least 1 hour; and / or the cryo-harvested cell product is stable at room temperature for at least 1, 2, 3, 4, 5, 6, 7, 8, 10, 15, 20, 24, 30, or 48 hours; and / or the cell product contains a solution comprising dextrose, one or more electrolytes, albumin, dextran, and / or DMSO; and / or The cell product is in a solution that is sterile, non-purulent, and isotonic. The method of claim 1.

9. the TARGET cell product can be used to treat the patient; the patient has cancer, and / or the patient has a viral, bacterial, fungal or parasitic infection, and / or the patient has a disease or condition involving inflammation, in some embodiments excluding cancer; and / or the patient has an autoimmune disease or condition, and / or the TARGET cell product is allogeneic to the patient, and / or the patient shows no signs of rejection or depletion of the TARGET cells, and / or Some methods of treatment further include administering to the patient a stimulatory agent that activates TARGET cells or an agent that induces a suicide gene. The method of claim 1.

10. 1. A method for generating monoclonal TCR-armed genetically engineered T (TARGET) cells using pluripotent stem cells (PSCs), comprising: (a) introducing into a TARGET cell a selected monoclonal T cell receptor (TCR) gene as the endogenous TCR gene when a T cell reprogrammed induced PSC (T-iPSC) line is used to generate said TARGET cell; or (b) introducing into the TARGET cells a selected monoclonal TCR gene as an exogenous TCR transgene if a non-T-iPSC PSC line is used to generate said TARGET cells; or / and differentiating the PSC cells of (a) or (b) to generate the monoclonal TCR-armed genetically engineered T (TARGET) cells. A method comprising:

11. 11. The method of claim 10, wherein the genetic modifications are performed according to an all-in-one manipulation methodology, which involves performing all genetic modifications on a master PSC line.

12. The method comprises: (a) placing the pluripotent stem cells in a serum-free, feeder-free PSC culture medium comprising at least one of bFGF, TGFβ, FLT3L, Noggin, Activin, and Bio for at least 12 hours; (b) combining the pluripotent stem cells from (a) with the at least one exogenous nucleic acid molecule disposed in an expression vector, culturing the PSC cells for at least 12 hours, and identifying pluripotent stem cells transduced with the expression vector; (c) placing the expression vector-transduced pluripotent stem cells from (b) dissociated into single cells in serum-free, feeder-free HSC differentiation culture medium A containing at least one of glutamax, ascorbic acid, monothioglycerol, insulin-transferrin-selenium, activin A, BMP-4, bFGF, VEGF, SB431542, CHIR99021, and a ROCK inhibitor for at least 12 hours, so that the cells form embryonic bodies; (d) placing the embryonic bodies of (c) in serum-free, feeder-free PSC differentiation culture medium B containing at least one of BMP-4, FGF, SCF, TPO, FLT3L, IL-6, IL-11, IGF-1, SB203580, and EPO for at least 6 days to form CD34+ hematopoietic stem cells; (e) CD34 obtained from (d) ＋ collecting and / or enriching hematopoietic stem cells; (f) the CD34 obtained from (e); ＋ placing the hematopoietic stem cells in a serum-free, feeder-free TARGET expansion cell culture medium containing at least one of serum albumin, recombinant human insulin, human transferrin, 2-mercaptoethanol, SCF, TPO, IL-3, IL-6, Flt3 ligand, human LDL, and UM171 for at least three days; (g) the CD34 obtained from (f) ＋ Placing the hematopoietic stem cells in a serum-free, feeder-free cell TARGET maturation medium containing at least one of serum albumin, recombinant human insulin, human transferrin, 2-mercaptoethanol, SCF, TPO, IL-3, IL-6, IL-7, IL-15, Flt3 ligand, and ascorbic acid; 11. The method of claim 10, wherein the monoclonal TCR-armed genetically engineered T (TARGET) cells are generated.

13. 13. The method of claim 12, further comprising placing the monoclonal TCR-armed genetically engineered T (TARGET) cells in target cell growth medium containing at least one TCR-cognate antigen or a non-specific TCR stimulatory reagent.

14. 13. The method of claim 12, wherein the method generates a TARGET cell that expresses at least 2,000 exogenous TCR polypeptides on the surface of the cell.

15. the exogenous nucleic acid molecule Alpha beta TCR, gamma delta TCR, Invariant NKT (TARGET) TCR, a non-invariant NKT TCR, and Mucosal-associated invariant TCR The method of claim 10, wherein the T cell receptor is encoded by at least one T cell receptor selected from the group consisting of:

16. 11. The method of claim 10, wherein the exogenous nucleic acid molecule encoding a T cell receptor comprises a promoter selected for its ability to resist silencing in the TARGET cell.

17. The method of claim 13 , wherein the promoter is a human ubiquitin promoter.

18. the exogenous nucleic acid molecule is contained in a lentiviral expression vector, and / or the exogenous nucleic acid molecule further encodes a polypeptide that stimulates T cells, a polypeptide that blocks T cell inhibitory factors, and / or a polypeptide that comprises an additional receptor; The method of claim 10.

19. 19. The method of claim 18, wherein the polypeptide comprises at least one of a chimeric antigen receptor (CAR), IL-2, IL-7, IL-15, IFN-γ, TNF-α, CD28, 4-1BB, OX40, ICOS, and FOXP3.

20. The method of claim 11 , wherein the genetic modification is performed before placing the PSC cells in differentiation medium.

21. 12. The method of claim 11, wherein the genetic modification is performed after placing the PSC cells in a differentiation medium.

22. 12. A monoclonal TCR-armed genetically engineered T (TARGET) cell produced by the method of claim 11.

23. 13. A monoclonal TCR-armed genetically engineered T (TARGET) cell produced by the method of claim 12.

24. The monoclonal target cell of claim 22 , wherein the target cell expresses CD4.

25. The monoclonal target cell of claim 23 , wherein the target cell expresses CD4.

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